In 2011, we published a paper overviewing the known pharmacokinetics, bioavailability, efficacy, and regulatory status of various purported "forms" of creatine "marketed with claims of improved physical, chemical, and physiological properties in comparison to CrM" [25]. The article concluded that "the efficacy, safety, and regulatory status of most of the newer forms of creatine found in dietary supplements have not been well-established" and "there is little to no evidence supporting marketing claims that these newer forms of creatine are more stable, digested faster, more effective in increasing muscle creatine levels, and/or associated with fewer side effects than CrM" [25]. Similarly, in its most recent position stand on creatine supplementation, the International Society of Sport Nutrition (ISSN) concluded: (1) "Creatine monohydrate is the most extensively studied and clinically effective form of creatine for use in nutritional supplements in terms of muscle uptake and ability to increase high-intensity exercise capacity"; (2) "Claims that different forms of creatine are degraded to a lesser degree than creatine monohydrate in vivo or result in a greater uptake to muscle are currently unfounded [25]"; and, (3) "Clinical evidence has not demonstrated that different forms of creatine such as creatine citrate [22], creatine serum [26], creatine ethyl ester [27], buffered forms of creatine [28], or creatine nitrate [29] promote greater creatine retention than creatine monohydrate [25]." These conclusions were reiterated more recently in a review related to myths and misconceptions about creatine

supplementation noting "there are no peer-reviewed published papers showing that the ingestion of equal amounts of creatine salts [30–33] or other forms of creatine like effervescent creatine [22], creatine ethyl ester [27,34,35], buffered creatine [28], creatine nitrate [29,36], creatine dipeptides, or the micro amounts of creatine contained in creatine serum [26,37,38] and beverages (e.g., 25–50 mg) increases creatine storage in muscle to a greater degree than creatine monohydrate [25]. Most studies show that ingestion of these other forms have less physiological impact than creatine monohydrate on intramuscular creatine stores and/or performance and that any performance differences were more related to other nutrients that creatine is bound to or co-ingested within supplement formulations."

Despite this clear scientific evidence, guidance from professional organizations, and regulatory challenges of selling other forms of creatine in the global marketplace, companies continue to market "new forms" of creatine that are purportedly more stable, bioavailable, and/or effective than CrM. Some have argued that mentioning some of the forms of creatine described in our 2011 paper served as validation or an endorsement that these forms of creatine were valid sources of creatine rather than the intended conclusion that these purported forms of creatine were not scientifically validated, safe, effective, and regulatory approved sources of creatine for dietary supplements. The intent of this comprehensive review is to provide an update regarding (1) how creatine is absorbed from food and/or dietary supplements into the body; (2) whether sources of creatine currently marketed and/or used in dietary supplements are bioavailable sources of creatine; and (3) whether any of these purportedly alternate forms of creatine are as effective in increasing creatine stores in the body to a greater degree than CrM. Based on this assessment, compounds commonly marketed as sources of creatine will be categorized as: (1) Strong evidence to support bioavailability, efficacy, and safety; (2) limited evidence to support bioavailability, efficacy, and safety; or (3) no evidence to support bioavailability, efficacy, and safety.

#### **2. Methods**

This paper was conducted as a systematic review of the literature related to sources of creatine marketed as ingredients for dietary supplements or found in dietary supplements, food products, and/or beverages marketed as containing creatine. This was accomplished by performing a PubMed search of the US National Library of Medicine database of different sources of creatine found in dietary supplements related to solubility, stability, bioavailability, supplementation, and performance. In addition, we conducted patent searches of the United States Patent and Trademark Office, the European Patent Office, the Japan Patent Office, and World Intellectual Property Organization (WIPO) mainly when no articles were found from a PubMed search on the purported form of creatine. We also reviewed company websites to assess claims and studies they cited to support claims, as well as publicly available reports of studies submitted as evidence to the court in cases related to these purported forms of creatine. To assess the current regulatory status, we conducted a search of key markets around the world, including the US Food and Drug Administration (FDA); the Therapeutic Goods Administration in the Department of Health of Australia; Health Canada; the State Administration for Market Regulation of China; the European Union Commission; the Japan Ministry of Health, Labor and Welfare; and the Korea Food and Drug Administration. The information obtained from this search was used to assess the legal and regulatory status related to the sale of purported forms of creatine in these marketplaces. Figure 1 shows a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart [39].

**Figure 1.** PRISMA flow chart.

#### **3. Bioavailability**

Bioavailability refers to the degree or rate at which a drug or substance is absorbed into the body, reaches the intended target site, and is available to influence physiological activity [40]. In terms of nutrients, bioavailability refers to the amount of the nutrient contained in the food or supplement that is delivered to the target tissue and available in the intended tissue for metabolic activity [40]. If food or supplement contains a large number of nutrients, but only a small percentage of the nutrient is liberated from the food or supplement, transported through the blood to the tissue, and ultimately taken up by the tissue, it is not very bioavailable. Similarly, if a similar quantity of food or supplement delivers less of the active nutrient to the target tissue than another food or supplement of equal quantity, it is comparatively less bioavailable. In the case of creatine, individuals who consume meat and fish in their diet typically have a plasma creatine level of around 25 μmol/L (about 3.75 μg/mL), and a muscle creatine content of about 120 mmol/kg dry muscle mass (DMM) [11,12,19,20]. Muscle creatine levels are typically lower in individuals following a vegan diet [41,42] and the elderly, who may not consume as much protein in their diet or have more difficulty digesting dietary protein [43,44]. Plasma creatine levels

increase after consuming creatine-containing food and dietary supplements containing a bioavailable source of creatine in proportion to the amount creatine ingested and digestion rate [2,41,42,45,46].

For a dietary source of creatine to be bioavailable, creatine must be: (1) absorbed as creatine into the blood and transported to tissues [2,25]; (2) transported into tissue via tissuespecific creatine transporter genes (e.g., CRT1 or SLC6A8 in muscle) [6,7]; and (3) increase tissue and cellular creatine and PCr content by a physiologically meaningful amount to influence metabolic activity (e.g., 20–40% in muscle and 10–20% in brain) [5,12,15,20,24,47]. In creatine research, the efficacy of creatine supplementation is determined by assessing the magnitude in which creatine supplementation protocol increases muscle creatine content as typically measured from muscle biopsy samples and/or muscle and brain creatine content as determined from magnetic resonance spectroscopy (MRS) [15]. Since oral ingestion of CrM is nearly 100% bioavailable (i.e., it's either absorbed by tissue or excreted in urine), whole-body creatine retention with CrM supplementation can also be estimated as the difference between daily intake of CrM and urinary creatine output [22]. Purported forms of creatine that do not increase blood creatine concentrations, do not increase uptake of creatine through tissue-specific creatine transporters, and ultimately do not increase tissue creatine levels by physiologically meaningful amounts would not affect creatine-related metabolic function. This is regardless of whether the purported form of creatine is more solubility in water, is more stable under various temperature and pH conditions outside of the body, or is delivered in food, gel, and liquid sources, as will be discussed below.

In support of this contention, classic experiments by Harris et al. [12] indicated that a dose of orally ingested creatine should ideally increase plasma creatine levels to greater than 500 μmol/L (75 μg/mL) to optimize tissue uptake. They reported that oral ingestion of 1 g or less of CrM had negligible effects on blood creatine content (i.e., rarely exceeding 100 μmol/L (15 μg/mL)). However, ingestion of one oral dose of 5 g of CrM (equivalent to about 1.1 kg of uncooked beef) resulted in plasma creatine levels of about 800 μmol/L (120 μg/mL) after 1 h of ingestion. It sustained plasma creatine above 200 μmol/L (30 μg/mL) for over 4–5 h (see Figure 2A). Supplementation of 5 g of CrM every 2 h maintained peak plasma creatine to levels exceeding 1000 μmol/L (150 μg/mL). Moreover, ingesting 5 g of CrM, 4 to 6 times a daily for 2-days or more significantly increased muscle creatine content of about 35%. Creatine uptake into the muscle was greatest during the first two days of CrM supplementation and declined over the next few days as muscle creatine levels became fully saturated. Subsequent studies from Hultman and colleagues [11] investigated the effects of various CrM supplementation strategies on changes in muscle creatine content. These experiments revealed that: (1) consuming 4 × 5 g of CrM per day for 6-days (i.e., creatine loading strategy) significantly increased muscle free creatine content by 33% and returned to baseline within 4-weeks after supplementation; (2) ingesting 4 × 5-g doses of CrM for 6-days followed by ingestion of 2 g/day of CrM for 28 days maintained a 36% increase in muscle creatine levels; and (3) ingesting 3 g/day of CrM for 35 days (i.e., low dose supplementation strategy) resulted in a gradual 16.7% increase in muscle creatine content. About 70% of the increase in the total creatine pool was observed in changes in free creatine content in the muscle.

Harris and associates [45] also conducted two experiments assessing the bioavailability of CrM in solution compared to creatine obtained from meat, crushed lozenges, and suspended in gel (see Figure 2B,C). In the first study, the researchers reported that ingestion of 2.5 g of CrM in solution (providing about 2.2 g of creatine) promoted a more rapid and greater increase in peak plasma creatine (287 ± 115 μmol/L or 42 μg/mL) than ingesting 408 g of lightly cooked steak containing 5.4 g (182 ± 52 μmol/L or 27 μg/mL). However, ingesting 5.4 g of creatine in lightly cooked meat promoted a more sustained increase in plasma creatine. Nevertheless, both strategies resulted in similar increases in area under the curve (AUC) values (507 ± 205 and 518 ± 153 μmol/h/L, respectively). In the second study, the researchers reported that ingestion of 2 g of CrM in solution resulted in peak plasma creatine levels of 386 ± 88 μmol/L (57 μg/mL) with an AUC of

622 ± 193 μmol/h/L. This compared to the peak value of 269 ± 67 μmol/L (41 μg/mL) with an AUC of 399 ± 196 μmol/h/L when CrM was administered in suspended gel and a peak creatine level of 277 ± 53 μmol/L and an AUC of 438 ± 131 μmol/h/L when CrM was administered as crushed lozenges. Collectively, these findings and are important because they demonstrate: (1) ingestion of 5 g of creatine from meat or 2–2.5 g of CrM administered in fluid, gels, and solids increased plasma creatine content by physiologically significant amounts needed to promote creatine uptake into the muscle; (2) the optimal single dose of CrM to increase plasma creatine levels is 5 g, but that ingesting 2–3 g will increase plasma creatine to sufficient levels to promote creatine uptake to tissue; (3) ingesting 5 g of CrM, 4 to 6 times a day for as little as 2 days was sufficient to significantly increase muscle creatine levels; (4) ingesting 5 g of CrM, 4 times a day for 6 days (i.e., 120 g total) increased muscle free creatine by about 35%; (5) consuming 3 g/day of CrM for 35 days (i.e., 105 g total) promoted a gradual 16.7% increase in muscle creatine; and (6) they provided a scientific basis for CrM supplementation recommendations and data for comparison of the efficacy of other purported forms of creatine marketed in dietary supplements [15].

**Figure 2.** Changes in plasma creatine concentrations after administration of 5 g of creatine monohydrate (CrM) in solution (**A**) [12]; water, 2 g of CrM administered in solution, or 408 g of slightly cooked meat containing 5.4 g of creatine (**B**) [45]; or 2 g of CrM provided in solution, gel suspension, or in a hard candy lozenge (**C**) [45].

#### *3.1. Methods to Assess Bioavailability*

#### 3.1.1. Assess Chemical Structure

The first step in assessing the bioavailability of a purported novel form of creatine is to determine whether the purported source of creatine contains a creatine molecule. Although this seems obvious, as will be seen below, some purported sources of creatine do not contain the complete creatine molecule. Instead, they only contain a portion of the creatine molecule structure or rearrange the chemical structure such that the compound is not really creatine. Moreover, some purported sources of creatine bind or complex compounds to creatine (or a portion of its structure) with bonds so strong (e.g., amide bonds) they would not likely break down into creatine through normal digestion, and therefore would not likely increase creatine levels in the blood or tissues. In other words, they are simply not bioavailable sources of creatine. Consequently, assessing the chemical structure of a purported form of creatine and whether associated bonds would easily disassociate into a creatine molecule is the first question that should be asked when evaluating claims about a purported "novel form" of creatine.

#### 3.1.2. Assess Changes in Blood Creatine Content

The next step is to determine if the purported source of creatine is absorbed through normal digestive processes into the blood and increases plasma creatine levels in a physiologically significant manner. Purported forms of creatine that do not significantly increase plasma creatine above normal fasting levels (i.e., about 25 μmol/L or 3.75 μg/mL) would have no effect on muscle creatine content because it would not deliver any creatine to tissues for uptake by creatine transporters. Likewise, sources of creatine that increase plasma creatine levels by less than 100 μmol/L (15 μg/mL) would not be considered viable sources of creatine for dietary supplements because they would not deliver enough creatine to target tissue to significantly increase creatine content [12,45]. Viable sources of creatine in dietary supplements should increase plasma creatine levels above 200–500 μmol/L (30–75 μg/mL) within the first hour of oral ingestion and promote a large increase in the AUC of creatine over a 4-to-5 h period [11,12,45]. However, an increase in plasma creatine alone does not provide definitive proof that a purported source of creatine is bioavailable or effective. Differences in plasma creatine after ingestion of a bioavailable source of creatine only suggests that absorption rates may differ. Higher blood creatine could mean that the source is not taken up as quickly into tissue, while lower levels could mean that less appears in the blood or creatine absorption into tissue is faster [48]. Ultimately, the source of creatine must be transported into tissues by creatine-specific transporters and increase tissue levels of creatine in physiologically significant amounts to affect creatine-related metabolism (i.e., 20–40%). Thus, it cannot be assumed that a purported source of creatine will be effectively transported into muscle based on solubility properties in fluid and/or changes in blood concentrations alone because sources of creatine that are not effectively transported into tissue could have higher plasma levels than those that promote a more rapid transport into the tissue. Consequently, it is important to assess the difference between arterial content (amount of creatine delivered to tissue) and venous creatine content or measure the amount of creatine retained in tissues directly to determine the amount of orally ingested creatine taken up by tissue.

#### 3.1.3. Assess Changes in Tissue Creatine Content

Thus, the third step in verifying the bioavailability of a purportedly novel source of creatine is to directly assess the effects of oral ingestion at recommended dosages on tissue creatine content. This is most often done by determining changes in muscle creatine content since 95% of creatine is stored in skeletal muscle. So-called forms of creatine that have been marketed as creatine but do not have any data showing the source of creatine increases muscle creatine content in humans should not be considered a viable source of creatine until such data are available. Purported sources of creatine that increase blood levels of creatine but do not increase tissue levels of creatine in physiologically effective amounts (i.e., 20–40% in muscle and 10–20% in brain) are not bioavailable sources of creatine. Purported sources of creatine that do not deliver similar increases in muscle creatine content than equivalent doses of CrM are less bioavailable sources of creatine than CrM. Moreover, purported derivatives or analogs of creatine that have no measurable effects on plasma creatine levels and do not increase muscle creatine content are not bioavailable sources of creatine, and therefore could have no physiological effects that have been reported in the literature from CrM supplementation. Likewise, if a form of creatine does not significantly increase muscle and/or brain creatine content in humans at the recommended dosages, it should not be considered a viable source of creatine for a dietary supplement. This includes making claims that lower doses of a purportedly more bioavailable source of creatine (e.g., 1–2 g) or "sprinkling" physiologically insignificant amounts of CrM or other purported creatine sources, derivatives, or analogs of creatine in supplements or beverages (e.g., 25–250 mg) promote similar or better benefits as CrM loading (e.g., 4 × 5 g/day for 5–7 days) or long-term supplementation (e.g., 3 g/day).

#### **4. Physio-Chemical Properties**

Figure 3 shows the structure of creatine and CrM. Creatine monohydrate was the first source of creatine marketed as a dietary supplement and remains the most common source of creatine found in dietary supplements [25]. Creatine monohydrate is considered the gold standard to compare other purported sources of creatine because of its well-known physiochemical properties, high bioavailability, stability, low cost, and a large number of studies that have demonstrated efficacy and safety [15,25]. CrM has been so extensively studied compared to other purported forms of creatine that when creatine supplementation is discussed in the literature, it is understood the authors are referring to CrM unless otherwise specified [15,17,18,49]. Nevertheless, CrM is formed by crystallization with water forming monoclinic prisms that hold one molecule of water per molecule of creatine [25]. This provides a powder containing 87.9% creatine that readily dissociates into creatine and water upon oral ingestion. The water in CrM can also be removed when exposed to heat at about 100 ◦C yielding anhydrous creatine that is 100% creatine [25]. However, due to the increased temperature used during the drying, anhydrous creatine contains higher amounts of the impurity creatinine. Creatine appears as internal salt and is considered a fairly weak base (pKb 11.02 at 25 ◦C) that forms salts with strong acids (i.e., pKa < 3.98) [25]. Creatine can also complex with other compounds via ionic binding (i.e., the attraction of positive cation and negative anion charges).

**Figure 3.** Chemical structure of creatine and creatine monohydrate.

Pischel and Gastner [50] described the basic process of industrial synthesis of CrM. The process involves adding acetic acid to an aqueous sodium sarconsinate solution and stirring to a pH value of about 10 and a temperature of about 80 ◦C. An aqueous cyanamide solution is then added to the medium and agitated to facilitate the reaction. After cooling, the crystalline CrM is filtered, separated, and then dried [50]. Creatine monohydrate is manufactured by using water as solvent in Germany has produced 99.9% pure CrM with no contaminants under the brand name Creapure®. Other sources of CrM, particularly from China, have been reported to contain contaminates like dicyandiamide, dihydrotriazine, dimethyl sulphate, thiourea, creatinine, and/or higher concentrations of heavy metals like mercury and lead due to the use of different chemical precursors, poorly controlled synthesis processes, using organic solvents, and/or less than adequate filtration methods that increase production of these contaminants [50]. For this reason, creatine monohydrate manufactured by AlzChem in Germany is considered the gold standard of creatine and has been the primary source of creatine used in hundreds of clinical trials conducted on CrM to establish safety and efficacy [15,25].

#### **5. Stability**

Creatine monohydrate is very stable in powder form, showing no signs of degradation to creatinine over years, even at elevated temperatures [25]. For example, Jäger [51] reported that CrM powder showed no signs of degrading to creatinine even with temperatures up to 40 ◦C (104 ◦F) for more than three years. Additionally, even when CrM was stored at 60 ◦C (140 ◦F), creatinine could only be detected in trace amounts after 44 months of storage [51]. However, creatine is not as stable in solution due to intramolecular cyclization that converts

creatine to creatinine (see Figure 4A). Generally, creatine degrades to creatinine in solution at a faster rate as pH decreases and temperature increases [25,52–54]. For example, as seen in Figure 4B, Harris and coworkers [55] reported that creatine is relatively stable for 3 days in solution at neutral pH (6.5 to 7.5) However, the rate of degradation to creatinine increased when stored at 25 ◦C when pH decreased (e.g., 4% at 5.5 pH; 12% at 4.5 pH; and 21% at 3.5 pH) [55]. However, as described below, the conversion of creatine to creatinine is halted at pH levels < 2.5. For this reason, it is recommended that CrM should be consumed immediately after it is mixed in an acidic beverage or refrigerated to slow the degradation to creatinine and consumed within a couple of days. However, recent reports presented shelf-life stability data of CrM suspended in a solution of 70% for 13-months at neutral pH and 100% for 12 months at a pH of 2.8 [56,57].

**Figure 4.** Conversion of creatine to creatinine (**A**) and influence of pH on creatine stability in solution (**B**). Creatine stability figure adapted from Howard and Harris [55].

As mentioned above, the degradation of creatine can be limited or prevented when creatine is in very low or very high pH environments [25]. In this regard, a pH > 12.1 promotes the deprotonation of the acid group. This makes it more difficult for intramolecular cyclization to form creatinine [25]. On the other hand, when pH is <2.5, the amide functional group on the creatine molecule is protonated and prevents the intramolecular cyclization (see Figure 5) [25]. Since stomach acid is generally less than 2.5, less than 1% of CrM is degraded to creatinine during digestion and 99% of creatine is taken up by tissue or excreted in urine after ingestion [12,25,58,59].

**Figure 5.** Influence of pH on creatine stability in solution.

#### **6. Solubility**

One of the limitations in terms of developing consumer products containing CrM is that CrM powder is not highly soluble. For example, when mixing CrM in solution, some CrM residue remains at the bottom of the glass requiring consumers to add more fluid, swirl, and quickly ingest to ensure they consumed all the creatine. While this has no effect on creatine bioavailability as CrM is nearly 100% bioavailable [12,25,58,59], there has been interest in finding ways to improve the solubility of creatine. The solubility of creatine in water increases linearly with increasing temperature. In this regard, about 6 g of creatine dissolves in one liter of water at 4 ◦C (39.2 ◦F) while 14 g/L are dissolved at 20 ◦C (68 ◦F), 34 g/L are dissolved at 50 ◦C (122 ◦F); and, 45 g/L are dissolved at 60 ◦C (140 ◦F) [25]. This is the reason that some researchers initially administered CrM to participants in warm to hot water [12] or hot tea [60]. Creatine solubility can also be improved by administering CrM in lower pH solutions like juices and sport drinks that generally have pH levels ranging from 2.5–3.5 [61] and/or blending CrM with carbohydrate and/or protein powders or in juice which helps suspend CrM in solution, reduce sedimentation, and enhance creatine uptake into muscle [20–23,62].

Dissolving creatine in more acidic environment is also the rationale in providing creatine in the form of easily disassociated creatine salts. Adding an acidic moiety to a creatine molecule lowers the pH of the water. For example, adding tri-creatine citrate to water yields a pH of 3.2 and increases solubility to 29 g/L whereas adding creatine pyruvate to water yields a pH of 2.6 and increases solubility to 54 g/L [63]. Creatine hydrochloride (HCl) has also been marketed as a pH lowering source of creatine with greater solubility than CrM [64,65]. While lowering pH and/or mixing creatine salts into solution enhances solubility, the amount of creatine contained in these forms of creatine salt must be equalized to CrM to deliver the same amount of creatine to the blood and tissues. In this regard, CrM contains 87.9% creatine whereas creatine citrate (40.6%), di-creatine citrate (57.7%), creatine pyruvate (59.8%), and creatine HCl (78.2%) contain less creatine by weight compared to CrM. Therefore, one would need to mix 1.54, 1.34, 1.32, and 1.11 times more of these forms of creatine in solution to deliver the same amount of creatine than CrM. Additionally, while mixing CrM in common juices and beverages with pHs ranging from 2.5–3.5 [61] would enhance solubility, it would also promote the conversion of creatine to creatinine over time [66]. Therefore, it is best to consume creatine salts or CrM with acid beverages soon after it is mixed so that the conversion of creatine to creatinine would be halted upon entering a more acidic environment in the stomach.

With that said, the only real advantage of mixing CrM in an acidic beverage is that it would leave less CrM in crystalized form at the bottom of a cup to swirl and consume during the last drink. If an individual consumes all the CrM (soluble or not), it will be bioavailability in terms of intestinal absorption, transport of creatine in the blood, transport of creatine through tissue-specific creatine transporters, and uptake of creatine into tissues. Similarly, if a bioavailable source of creatine is consumed at physiologically effective doses, it is not degraded during digestion, and it increases blood and tissue creatine content by physiologically meaningful amounts (i.e., 20–40%), it does not matter whether a form of creatine has better mixing characteristics and/or is more soluble. Research has clearly shown that CrM is not degraded into creatinine during normal digestion, it is nearly 100% bioavailable [12,25,58,59], and markedly increases blood and tissue creatine content. There are no data showing that any other purported form of creatine is more effective in increasing tissue creatine content than CrM [62]. Therefore, claims that a given form of creatine is more effectively absorbed than CrM because it is more soluble in water is unsupported marketing hyperbole.

#### **7. Purported Creatine Related Compounds**

Table 1 provides a list of creatine-related compounds that are listed in PubChem when doing a search on the word creatine. As of this writing, 88 creatine-related compounds are listed in the PubChem database. A few others that have been mentioned in patents, published literature, or on international regulatory authorities' lists are also mentioned at the bottom of Table 1. While not all these compounds have been used in dietary supplements or assessed for bioavailability, it provides an overview of the types of creatine-related compounds that have been developed. Interestingly, some creatine derivatives and analogs that have been marketed as bioavailable sources of creatine in dietary supplements are not listed in this database because the molecular structures have been altered such that the compound does not contain the creatine molecule. Consequently, they should not be considered a bioavailable source of creatine unless studies show that it increases creatine levels in the blood and target tissue (i.e., muscle and brain). This view is consistent with

the United States FDA definition that a dietary supplement is considered a new dietary ingredient (NDI) if the ingredient has been chemically altered from its natural form [67].

**Table 1.** Theoretical creatine content of purported sources of creatine-related compounds in the PubChem database based on molecular weight.

Adapted from Jaeger et al. [25]. MW represents molecular weight. † represents theoretical creatine content based on the MW of creatine assuming a full molecule of creatine is in the compound, and it is liberated as creatine from the compound. Many sources have not been studied to verify ingestion increases blood levels of creatine or tissue creatine content. Therefore, listing a compound in this table does not validate if the marketed source contains creatine, is bioavailable as creatine, or is an effective source of creatine. Not listed on PubChem as a creatine-containing compound: creatine maleate, creatine fumarate, creatylglycine ethyl ester fumarate, polyethylene glycosylated creatine, polyethylene glycosylated creatine HCL, Creatine Serum, creatyl-L-leucine.

It should be noted that different creatine-related compounds shown in Table 1 contain less creatine by molecular weight than CrM, assuming that a full creatine molecule is contained in the compound and would be liberated as creatine in circulation. Consequently, it would take a greater amount of most of these sources of creatine in a dietary supplement to provide equivalent amounts of creatine delivered from CrM if the other sources were

indeed bioavailable. For example, creatine citrate (CC) contains 53.9% less creatine by molecular weight than CrM. Therefore, a supplement would need to provide 9.3 g of CC to equal a typical supplementation dose of 5 g of CrM if it had similar bioavailability. Creatine ethyl ester (CEE) has only 6.3% less creatine by molecular weight than CrM. However, as will be discussed below, some of the creatine in CEE converts to creatinine during digestion and therefore it is less bioavailable than CrM. Therefore, more CEE would have to be provided in a supplement to provide an equivalent amount of creatine to tissue and it may increase serum creatinine levels to a greater degree. Additionally, if a purported source or derivative of creatine does not break down and increase blood creatine levels and creatine content in muscle, it would not be a bioavailable source of creatine no matter how much was in the supplement. These are important points to consider when developing nutritional formulations or conducting research with these other sources of creatine.

#### **8. Strong Evidence to Support Bioavailability, Efficacy, and Safety** *Creatine Monohydrate*

As noted above, CrM is the gold standard to compare other purported forms of creatine due to its known bioavailability, pharmacokinetics, efficacy, and safety [5,14,15,25,62]. Prior studies indicate that CrM loading (i.e., 4 × 5 g/day for 5–7 days) or low-dose longterm intake (e.g., 3–6 g/day for 4–12 weeks) increases muscle creatine retention typically by 20–40% depending on initial creatine content in the muscle [12,22,68–71] and brain creatine content by 5–15% [72–77]. CrM supplementation has been reported to improve acute exercise performance particularly in intermittent high-intensity exercise bouts as well as enhance training adaptations in adolescents [78–82], young adults [29,55,83–92], and older individuals [8,77,93–101]. High-intensity exercise performance is generally increased by 10–20% with greater improvements seen in individuals starting the supplementation protocol with lower muscle creatine and PCr content [102]. Improvements in performance have been reported in individuals participating in weight training [55,89,95,103–113], running [114–118], soccer [87,119,120], swimming [79,80,121–124], volleyball [125], softball [126], ice hockey [127], golf [128], among others [24]. Men and women have been reported to benefit from CrM supplementation in populations ranging from children to elderly populations [47,80,119,126,129–133].

Uptake of creatine into muscle with CrM supplementation has been reported to be enhanced when CrM is consumed with carbohydrate [20–22] and carbohydrate and protein [23]. Co-ingesting CrM with nutrients that improve insulin sensitivity like Dpinitol [134], Russian Tarragon [135], and Fenugreek extract [136] have been reported to enhance creatine retention with limited to no additive effects on exercise performance or training adaptations [89,136,137]. There is also no evidence that supplementation of micronized versions of CrM (i.e., CrM with smaller mesh size particles) are more bioavailable than CrM with normal mesh sized particles [138–140] or consuming CrM in effervescent fluid promotes greater creatine retention or performance benefits [22,141]. This makes sense in that while small differences in creatine retention may enhance the rate of saturating creatine stores (e.g., in 4 versus 5 days) or ensure more consistent response to CrM supplementation (e.g., consuming creatine with carbohydrate and/or carbohydrate and protein), once creatine stores are fully saturated, there would be no ergogenic or training advantage. No side effects have been reported with CrM supplementation other than a desired weight gain [15]. Additionally, there is no convincing evidence that CrM causes common anecdotal myths like bloating, gastrointestinal distress, disproportionate increase in water retention, increased stress on the kidneys, increased susceptibility to injury, etc. [62,142–144]. Many of these claims have been described in marketing materials by companies attempting to gain market share for their purported creatine-containing products [25,62].

Based on a large body of evidence, the ISSN concluded that CrM is the most effective ergogenic nutrient currently available to athletes in terms of increasing high-intensity exercise capacity and lean body mass during training [8,15]. Position stands by the American Academy of Nutrition, Dietitians of Canada, and the American College of Sports Medicine

on nutrition and athletic performance [17,49]; the International Olympic Committee consensus statement on dietary supplements and the high-performance athlete [18]; and, the Office of Dietary Supplements, Dietary Supplement Fact Sheets on Dietary Supplements for Exercise and Athletic Performance [145] provided similar conclusions. Thus, there is consensus among professional organizations that CrM is an effective nutritional ergogenic aid that may benefit athletes involved in numerous sports and individuals initiating exercise training to promote health and fitness.

Given the metabolic and ergogenic properties of CrM [7,14,15,24], there has also been interest in assessing the effects of CrM supplementation in various clinical populations that may benefit from increasing high-energy phosphate availability and/or increasing strength and muscle mass [5]. A recent Special Issue on creatine supplementation for health and clinical diseases overviewed the metabolic basis of creatine in health and disease [5] and potential health and/or therapeutic benefits of CrM supplementation for pregnancy and newborn health [146], children and adolescents [147–151], physically active young adults [24], rehabilitation [152], women's health [133], older adults [44], brain health and cognitive function [74], glucose management and diabetes mellitus [153], immunity [154], T cell antitumor immunity and cancer therapy [155], heart health [156], vascular health [157], inflammatory bowel disease [158], chronic renal disease management [159], and post-viral fatigue [160]. From what we can see, all studies in these populations used CrM as the source of creatine. Thus, there is substantial evidence to support the safety and efficacy of CrM supplementation (see Table 2). Additionally, this body of evidence provides the basis to compare the efficacy and safety of other purported sources of creatine. For this reason, CrM is classified as having strong evidence from pharmacokinetic and tissue bioavailability studies, numerous randomized and controlled clinical trials, and a long-history of safety assessed in clinical trials and historical widespread use supporting bioavailability, efficacy, and safety.

**Table 2.** Example of studies showing bioavailability, efficacy, and safety of CrM supplementation.

**Table 2.** *Cont.*

R = randomized; DB = double-blind; *p* = placebo; SB = single blind; C = crossover, CrM = creatine monohydrate, PCr = phosphocreatine; TCr = total creatine; Pi = inorganic phosphate; CHO = carbohydrate; PRO = protein; FFM = Fat-Free Mass, TAN = total adenine nucleotide pool; MVC = maximal voluntary contractions; α-LA = alpha lipoic acid; MRS = magnetic resonance spectroscopy; GI = gastrointestinal.

#### **9. Some Evidence to Support Bioavailability, Efficacy, and Safety**

#### *9.1. Creatine Salts*

Creatine salts were introduced into the marketplace in the early 1990s and are formed by adding an acid moiety to creatine, complexing an acid to the creatine molecule, or adding an acid to a complexation product [25]. The rationale was to combine creatine with acids that could easily dissociate (e.g., ionic bond) upon ingestion, thereby not only serving as a viable way to deliver creatine to tissue, but also deliver other nutrients that may have ergogenic properties and/or promote a synergistic metabolic effect with creatine. Additionally, to find ways to improve physical characteristics like solubility of creatine. To do so, the creatine salt must deliver physiologically effective doses of creatine (i.e., 3–5 g per serving) to the blood and tissue in an equivalent manner as CrM to be comparatively effective. Additionally, the acid added to creatine would have to provide a more synergistic effect than simply co-ingesting CrM with the acid independently in a nutritional formulation. Finally, the theoretically added benefit must justify the additional expense in producing the creatine salt and including it in a nutritional formulation.

A number of creatine salts have been marketed as sources of creatine for dietary supplements including creatine citrate (including di- and tri- forms) [172–174]; creatine maleate, creatine fumarate, creatine tartrate [31]; creatine pyruvate [32,174–176]; creatine ascorbate [33]; and creatine orotate [30,176,177], among others. Some creatine salts are less stable when compared to CrM. For example, storage of tri-creatine citrate at 40 ◦C (104 ◦F) for 28 days results in formation of 770 ppm of creatinine compared to no measurable amount with CrM powder [63]. Adding carbohydrate to the formulation has been reported to improve the stability of some creatine salts [178]. However, creatine salts would also have less stability than CrM in solution since adding the acid to creatine decreases pH to ranges that would promote greater formation of creatine to creatinine in solution over time. The following summarizes results of studies that provide some evidence of bioavailability, efficacy, and safety of creatine salts.

#### 9.1.1. Creatine Citrate

Figure 6 shows the chemical structure of two common creatine salts. Jäger et al. [63] compared the effects of oral ingestion of 5 g of CrM to 6.7 g of tri-creatine citrate (CC) and 7.7 g of creatine pyruvate (CPY) that provided equimolar amounts of creatine. Tri-creatine citrate is a 1:1 salt of creatine and citric acid, with two additional creatines forming a complex with the 1:1 salt. The second and third acid moiety of citric acid are not strong enough acids to form salts with creatine. Peak concentrations of creatine were significantly higher with CPY (CrM 761.9 ± 107.7, CC 855.3 ± 165.1, CPY 972.2 ± 184.1 μmol/L) while AUC values did not significantly differ among treatments (2384 ± 376.5, 2627 ± 506.8, 2985 ± 540.6 mM/h, respectively). Results support contentions that provision of equimolar amounts of CC and CPY can serve as a bioavailable source of creatine. Conversely, Gufford et al. [179] reported that CC and CPY had less permeability than CrM across caco-2 monolayers cells, which is used as a model to assess intestinal absorption. However, it is unlikely that the small differences in blood creatine levels observed would promote greater creatine retention and/or a bioenergetic advantage. To date, we are unaware of any study that has evaluated the effects of CC or CPY on muscle or brain creatine content.

**Figure 6.** Chemical structure of tri-creatine citrate and creatine pyruvate with plasma creatine changes after oral administration of equal molar doses of creatine monohydrate (CrM), creatine citrate (CC) and creatine pyruvate (CPY). Adapted from Jäger et al. [63].

In terms of efficacy, several studies have evaluated whether creatine citrate (CC) supplementation can affect exercise capacity. For example, Smith and colleagues [180] found ingesting 20 g/day of di-creatine citrate for 5 days delayed neuromuscular fatigue in women. Jäger et al. [174] reported that ingestion of 5 g/day of CC (providing 3.25 g/day of creatine) for 28 days significantly increased intermittent maximal effort handgrip force compared to placebo. Graef and coworkers [181] reported that 10 g/day of CC supplementation for 30 days during high-intensity interval training significantly increased the ventilatory

threshold, but did not enhance maximal aerobic capacity. Smith and colleagues [172] reported that 20 g/day of creatine di-citrate supplementation for 5 days had no detrimental or ergogenic effects on running critical velocity, aerobic capacity, or time to exhaustion. Finally, Fukuda and assistants [182] reported that CC supplementation (4 × 5 g with 18 g dextrose for 5 days) improved anaerobic run capacity in men, but not women. While these studies provide evidence that CC can serve as a viable source of creatine by increasing blood creatine level in a similar manner as CrM and there is some data supporting the ergogenic benefit compared to placebo, the impact of CC supplementation has not been assessed on muscle or brain creatine content. Therefore, there are no studies indicating that CC is more bioavailable, more effective, or a safer source of creatine than CrM. Given this, CC is categorized as having limited evidence to support bioavailability, efficacy, and safety.

#### 9.1.2. Creatine Pyruvate

Calcium pyruvate supplementation (e.g., 6–25 g/day) has been reported to affect exercise performance and promote fat loss [183–186]. Stone et al. [83] also reported that CrM and the combination of CrM and calcium pyruvate supplementation during 5 weeks of offseason training improved training and body composition adaptations. In contrast, calcium pyruvate supplementation alone had no effects. Therefore, there was some rationale in developing a creatine salt with pyruvate [32]. As seen in Figure 7, ingestion of 7.3 g of CPY promoted significant increases in plasma creatine levels in a similar manner as ingesting 5 g of CrM [63]. This group also reported that ingestion of 5 g/day of CPY (providing 3 g/day of creatine) for 28 days significantly increased intermittent maximal effort handgrip force [174]. Another study found that ingesting 7 g/day of CPY for 7 days had no effects on endurance capacity or repeated sprint performance in cyclists [175]. However, ingesting 7.5 g/day of CPY improved paddle rate and lowered blood lactate in Olympic canoeists suggesting an improvement in aerobic exercise efficiency [187]. These studies indicate that CPY can serve as effective source of creatine to increase blood creatine content and a few short-term studies suggest there may be some ergogenic values. However, since there are no data assessing the effect of CPY supplementation on muscle or brain creatine content and only a limited number of studies have evaluated efficacy and safety, CPY is classified in the limited bioavailability, efficacy, and safety category. With that said, there is no evidence that CPY is more effective than CrM in increasing muscle creatine content and/or performance.

**Figure 7.** Chemical structure of magnesium creatine.

#### *9.2. Magnesium Creatine Chelate*

Magnesium creatine chelate (MgCr-C) has been marketed as a more bioavailable source of creatine (see Figure 7). The rationale is that since magnesium is a cofactor in ATP reactions and the only mineral that decreases during exercise, there may be additive benefit in combining creatine with magnesium. There are also claims that MgCr-C supplementation can improve muscle protein synthesis. A patent described bioavailable chelates of creatine and essential metals [188]. Since MgCr-C contains about 84.4% of creatine by molecular weight (see Table 1), it could theoretically serve as a good source of creatine if creatine easily dissociates from MgCr-C and equimolar amounts of creatine were consumed compared to CrM. However, it is marketed as a much more bioavailable source of creatine than CrM with recommended doses of only 1 g per 18.2 kg (40 lbs.) of body weight per day (about 3.8 g/day for a 70 kg individual). We are aware of no data showing that MgCr-C increases blood creatine levels or promotes greater creatine retention in skeletal muscle. Thus, there are no data supporting that MgCr-C is more bioavailable to tissue than CrM.

Several studies have evaluated the effects of MgCr-C on performance related variables. For example, Brilla and coworkers [189] evaluated the effects of ingesting 5 g of CrM with 800 mg of magnesium oxide or magnesium plus 5 g of MgCr-C for 2 weeks compared to placebo on body water and isokinetic strength performance in recreationally active participants. Results revealed that body water increased with MgCr-C, but not CrM while torque and power increased similarly with CrM and MgCr-C. Selsby and colleagues [190] evaluated the effects of supplementing 2.5 g/day of CrM, MgCr-C, or a placebo for 10 days on strength and muscle endurance. Results revealed that CrM and MgCr-C were both effective in increasing performance with no differences observed between types of creatine ingested. Finally, Zajac et al. [191] reported that 5.5 g/day of MgCr-C supplementation during 16 weeks of soccer training improved repeated sprint ability performance compared to placebo. Interestingly, creatinine levels were also significantly increased throughout training with MgCr-C (0.83 to 1.87 mmol/L) compared to placebo (0.92 to 0.82 mmol/L), which is higher than reported in other long-term CrM studies during training in hot and humid environments that administered 5–10 g/day of CrM for 21 months in well-trained athletes [144]. However, this study did not compare the effects of consuming MgCr-C to CrM, and performance changes were consistent with other studies conducted on CrM supplementation. Consequently, there is no data showing that MgCr-C increases blood or muscle creatine and there is only limited data showing potential ergogenic effect. There is also no evidence that MgCr-C is more bioavailable, efficacious, and/or a safer source of creatine than CrM. For this reason, MgCr-C is listed in some evidence to support bioavailability, efficacy, and safety category.

#### *9.3. Creatine Ethyl Ester*

Another source of creatine that claims to have better solubility, bioavailability, and efficacy than CrM is creatine ethyl ester (CEE). CEE is basically a creatine molecule with a H+ removed from the second N position (i.e., NH versus NH2) and a methyl group (CH2- CH3) added to the terminal O position through an esterification reaction (see Figure 8). Thus, CEE is a chemical alteration of creatine and is not actually creatine. For CEE to act like creatine it would have to be de-ethylated and an H+ added back to the NH of the molecule at nearly 100% efficiency to deliver 94% of an equivalent CrM dose. Marketing claims suggest that CEE is absorbed faster and more efficiently than CrM, so no loading dose is needed. Additionally, CEE is claimed to have less anecdotal side effects than CrM like bloating and dehydration. For this reason, the recommended dosages of CEE are typically 2–6 g/day.

**Figure 8.** Chemical structure of creatine and creatine ethyl ester.

While proponents of CEE have assumed that all orally ingested CEE is converted to creatine in vivo, available studies suggest it is less efficient. For example, Childs and Tallon reported that CEE rapidly degrades to creatinine when exposed to stomach acid [34]. Giese et al. [35,192] reported that under physiological conditions, CEE non-enzymatically converted to creatinine with no measurable conversion to creatine (see Figure 9). Likewise, Katseres and colleagues [193] reported that the half-life of CEE was in the order of one minute suggesting CEE may hydrolyze too quickly to reach muscle cells in its ester form. On the other hand, Gufford et al. [194] reported that CEE converted to creatinine in a linear manner as pH levels dropped below 8.0 and that CEE was mostly stable at a pH of 1.0. Since acidity in the stomach generally ranges from 1.5 to 3.5, it is likely that some CEE is degraded into creatinine during normal digestion while delivering some level of creatine to blood.

**Figure 9.** Degradation of creatine ethyl ester to creatinine. Adapted from Jäger et al. [25].

Spillane and colleagues [27] compared the effects of supplementing the diet with a 0.30 g/kg of fat-free body mass (approximately 20 g/day) for 5 days followed by ingestion of 0.075 g/kg of fat free mass (approximately 5 g/day) for 42 days of a placebo, CrM, or CEE on muscle creatine content and performance adaptations. If CEE is bioavailable, those taking CEE should have increased muscle creatine content better than those taking a placebo. Likewise, if CEE was more bioavailable than CrM, greater changes would be seen in the CEE group compared to those in the CrM group. As seen in Figure 10A, fasting serum creatine levels significantly increased only in the CrM group. While this was not a pharmacokinetic oral dose study, it is interesting that CEE supplementation had no effect on fasting serum creatine levels compared to a placebo. Conversely, CEE significantly increased serum creatinine levels by more than two-fold after 6, 27, and 48 days of supplementation in comparison to the placebo and CrM groups (Figure 10B). The values observed exceeded normal creatine values even for highly trained athletes training in hot and humid environments [13]. In addition, while CEE supplementation promoted a significant increase in muscle total creatine content after 27 days of supplementation compared to those ingesting a placebo, those taking CrM observed significantly greater increases compared to the placebo and CEE groups (Figure 10C). These findings suggest a large amount of CEE is converted to creatinine and CEE is less effective in increasing muscle creatine content than CrM. This was despite including a 20 g/day loading dose of CEE that manufacturers claimed is unnecessary due to greater bioavailability. Moreover, CEE supplementation did not promote greater changes in body composition, strength, or anaerobic power during training compared to CrM supplementation. These findings directly refute claims that CEE is more bioavailable source of creatine than CrM and that CEE promotes greater training adaptations than CrM. Further, the clinically significant increase in creatinine levels observed should raise some concerns about potential safety of CEE as has reported two case studies [195,196].

**Figure 10.** Serum creatine (**A**), serum creatinine (**B**), and muscle creatine content (**C**). \* Represents significant change from baseline. † in panel A indicates significantly higher serum creatine concentrations in CrM when compared to PLA (*p* = 0.007) and CEE (*p* = 0.005). † in panel B indicates serum creatinine in the CEE group was greater than PLA (*p* = 0.001) and CrM (*p* = 0.001). † in panel C shows the PLA group was significantly less than the CrM (*p* = 0.026) and CEE (*p* = 0.041) groups. Adapted from Spillane et al. [27].

With that said, some have pointed to the results of a recent study conducted by Arazi and associates [197] to support claims about the efficacy of CEE. The researchers evaluated the effects of 6 weeks of CEE supplementation (20 g/day for 5 days and 5 g/day for 37 days) compared to consuming a placebo during resistance training (3 sets of 8–10 repetitions at 60–80% or one repetition maximum, 3 times per week) in untrained, younger, and underweight men. The investigators reported that CEE supplementation during resistancetraining promoted significant increases in body weight and leg press strength, while percent body fat decreased to a greater degree with placebo ingestion. In addition, some differences were reported in anabolic and catabolic hormones that prior have not been previously studies conducted on creatine had not reported [62]. While this study indicates that higher than recommended doses of CEE can positively affect training adaptations, this study did not assess the effects of CEE supplementation on blood or muscle creatine content or compare the efficacy of CEE supplementation to CrM. The results observed are consistent with those found in the Spillane et al. study [27] in that CEE had some benefit over placebo ingestion, but results were not better than CrM. Given that CEE promoted a modest but less effective increase in muscle creatine content in that study, one would expect some benefit of CEE supplementation during training if higher than recommended doses are ingested. However, there is no evidence that ingesting recommended doses of CEE is effective or that ingesting typical CrM loading, and maintenance doses of CEE is more effective than CrM. We are also not aware of any studies that have evaluated other marketed forms of CEE (i.e., creatine methyl ester hydrochloride, di-acetyl creatine ethyl ester, creatine ethyl ester pyruvate, creatine ethyl ester malate, or creatylglycine ethyl ester fumarate). Nevertheless, since there is some evidence that ingesting high doses of CEE can increase muscle creatine content and performance compared to placebo, we have categorized CEE in some evidence category. However, we recommend that additional research evaluate safety given the increased creatinine levels observed.

#### *9.4. Creatine HCl*

Creatine hydrochloride (Cr-HCl) has been marketed as a more bioavailable source of creatine than CrM. As shown in Figure 11, Cr-HCl is a salt of HCL and creatine molecule. Like other creatine salts, adding hydrochloric acid to creatine would be expected to decrease pH and improve solubility. Marketing claims indicate that Cr-HCl has a 38 times greater bioavailability than CrM [198]. The basis for this claim appears to come from a report from Gufford and colleagues [179] who conducted physiochemical characterization studies on several N-methylguanidinium salts, including creatine Cr-HCl. They reported that Cr-HCl contains about 78% creatine by molecular weight and that Cr-HCl was 37.9 times more soluble in water than CrM at 25 ◦C. However, CrM was assessed at a saturation pH of 8.6 while Cr-HCL was measured at a saturation pH of 0.3. While mixing creatine in

acidic solutions may improve solubility when mixed in water, as noted above, it would have no effect on bioavailability. These claims are also apparently based on a report from Alraddadi et al. [199] who conducted a bioavailability study in rats with labeled creatine (creatine-13C) at a low (10 mg/kg) and high (70 mg/kg) oral doses. They then assessed the amount of creatine-13C incorporated into plasma, muscle and brain tissue and used a simulated prediction model to estimate how Cr-HCl would theoretically affect tissue creatine retention based on differences in solubility. While this is an interesting approach, there are several problems with using these findings to make claims about Cr-HCl. First, this is only a theoretical modeling study. The researchers did not directly compare Cr-HCl to CrM intake on plasma or tissue creatine content. Second, it is well-known that there are species specific differences in creatine metabolism and storage [200]. Therefore, you cannot directly extrapolate results from mice or rat data to human creatine oral dosing studies. Pharmacokinetic and creatine retention studies need to be conducted in humans to assess whether Cr-HCl promotes greater creatine retention in tissue to assess the validity of this claim.

**Figure 11.** Comparison of creatine monohydrate and creatine HCl structures.

As of this writing, no PubMed indexed articles have been published on Cr-HCl and muscle creatine retention or performance. However, several articles have been published in non-indexed journals from Brazil that have been cited in marketing materials. In the first study, de França and colleagues [65] evaluated the effects of supplementing the diet with 1.5 g/day of Cr-HCl, 5 g/day of Cr-HCl, and 5 g/day of CrM compared to controls during 4 weeks of resistance-training in 40 young recreational weightlifters on strength gains and skinfold caliper determined body composition. The researchers reported some benefits of Cr-HCl and CrM supplementation on leg press and skinfold determine body composition. However, the use of skinfold calipers to estimate body composition and statistical analysis methods employed make it difficult to draw any conclusions. In fact, gains in fat-free mass were greatest in the CrM group (+1.7 kg), but supposedly not significantly different than observed with 5 g/day of Cr-HCL (+1.6 kg) that were reported to be significantly different than controls (+1.1 kg) and those ingesting 1.5 g/day of Cr-HCl (+1.1 kg). In a follow-up study [201], this research group administered 5 g/day of CrM or 1.5 g/day of Cr-HCL with 3.5 g/day of resistant starch for 30-days to Brazilian Olympic level athletes. Results revealed both groups increased skinfold caliper determined fat-free mass and strength, although bioelectric impedance determined total body water was increased to a greater degree in the CrM group (CrM + 1.81 L vs. Cr-HCL + 0.24 L). This would be expected, given the creatine content based on molecular weight in these dosages was 35.1 g in the Cr-HCl group compared to 131.9 g in the CrM group over the 30-day period. Finally, a study conducted by Tayebi and Arazi [202] evaluated the effects of ingesting 3 g/day of Cr-HCL, 3 g/day of CrM, and 20 g/day of CrM, or a placebo for 7 days on anaerobic power and hormone levels. Results revealed that ingestion of 3 g/day of Cr-HCl did not promote greater gains in performance or hormonal responses than 3 or 20 g/day of CrM as claimed. The authors concluded that Cr-HCl does not appear to be a more effective source of creatine than CrM. Thus, while Cr-HCl is a simple salt that should readily disassociate into creatine and HCL, there is no evidence that Cr-HCl is absorbed more effectively than CrM in humans; Cr-HCl promotes greater muscle creatine retention than CrM at equivalent

doses; or, that lower doses of Cr-HCl are as effective as standard supplementation protocols with CrM. Given this analysis, Cr-HCl is classified in some evidence category to support efficacy compared to placebo. However, claims that Cr-HCl is more bioavailable, effective, and/or a safer source of creatine than CrM are not supported.

#### *9.5. Creatine Nitrate*

A number of studies have indicated that dietary nitrates, typically ingested in the form of beet root juice or nitrate powder, can improve endurance [203] and explosive exercise capacity [204]. Recommended dosages generally range between 300–600 mg ingested 1–2 h prior to exercise [204–206]. Since nitrate can ionically bond to creatine and form a salt (see Figure 12), creatine nitrate (CrN) has been developed and marketed as a bioavailable source of creatine for dietary supplements [207]. By molecular weight, CrN contains 67.5% creatine. Therefore, ingesting 1 g of CrN would theoretically provide 0.675 g of creatine and 0.325 g of nitrate. Marketing claims suggest greater bioavailability and therefore recommended doses are typically 1–2 g of CrN per/day [208]. In terms of bioavailability, there is limited data available. However, Galvan and colleagues [29] conducted a pharmacokinetic study evaluating the effects of acute oral ingestion of a placebo, 1.5 g of CrN (CrN-Low), 3 g of CrN (CrN-High), and 5 g CrM on blood creatine and nitrate levels. Results revealed that the plasma creatine AUC over a 5 h period for CrM (5634.4 ± 1949.8 μmol/L) was significantly greater than the placebo (1012.4 ± 1882.2 μmol/L), CrN-Low (2342.0 ± 3133.3 μmol/L, *p* = 0.004), and CrN-High (1761.7 ± 3408.8 μmol/L, *p* = 0.007) treatments with no differences seen between the CrN dosages. Conversely, the nitrate AUCs in the CrN groups were significantly greater than the placebo and CrM treatments in a dose related manner. These investigators also evaluated the effects of ingesting four doses a day of either a placebo (5 g dextrose), CrM (3 g CRM with 2 g dextrose, CrN-Low (1.5 g CrN, 3.5 g dextrose), and CrN-High (3 g CrN, 2 g dextrose) for 7 days followed by ingesting one dose per day for 21 days as a maintenance dose. Muscle biopsies were obtained at 0, 7, and 28 days to assess muscle creatine content. Results revealed that 7 days of creatine loading (12 g/day of CrM and CrN) significantly increased muscle creatine content in the CrM (7.1 mmol/kg DW) and CrN-High (4.6 mmol/kg DW). However, no difference was seen compared to placebo was observed when ingesting 4 × 1.5 g/day of CrN for 7 days or after 28 days of taking 1.5 or 3 g/day of CrN. These findings suggest that CrN can be a bioavailable source of creatine proportional to the amount of creatine delivered during the loading phase (i.e., 54.6 g for CrN-High versus 73.8 g for CrM), but not more bioavailable than CrM when equivalent doses are ingested. On the other hand, Ostojic et al. [209] conducted a study evaluating the effects of CrM and CrN supplementation on MRS determined skeletal muscle creatine content and markers of health. In a randomized and crossover manner with a 7 day washout period, participants ingested a placebo, 3 g/day of CrN, 3 g/day of CrM, or 3 g/day of CrN + 3 g/day of CrM for 5 days. This theoretically provided a total of 0, 9.75, 13.2, and 22.9 g of creatine during the 5-day period. The researchers found that peak serum creatine increased to a greater degree with CrN + CrM supplementation (CrM 118.6 ± 12.9, CrN 163.8 ± 12.9; CrN + CrM 183.7 ± 15.5 μmol/L) while muscle creatine increased to a greater degree with CrN ingestion (CrM 2.1%, CrN 8.0%, CrN + CrM 9.6%). However, a limitation to this study is that only a 7-day washout period was observed between treatments. It is well known that it takes about 4 weeks for muscle creatine to return to normal after creatine supplementation [47]. Thus, it is possible that the testing order may have confounded results. Nevertheless, results are conflicting on whether shortor long-term CrN supplementation (3 g/day) significantly increase muscle creatine levels.

**Figure 12.** Chemical structure of creatine nitrate. (**A**,**B**) show 5 h area under the curve data after plasma creatine and plasma nitrate, respectively. (**C**) shows mean changes in muscle creatine content with 95% confidence intervals after 7 days of loading 4 doses/day and 21 days of ingesting 1 those/day. † Represents significant change from baseline, PLA and P represent placebo, CrM represents creatine monohydrate, and CrN represents creatine nitrate.

In terms of performance, Gavan and colleagues found some ergogenic benefit of CrN supplementation on muscular endurance, but they were unrelated to changes in muscle creatine content, suggesting the ergogenic benefit was primarily due to providing ergogenic levels of nitrate and not creatine at the dosages studied. Dalton and colleagues [36] reported that ingesting 3 and 6 g/day of CrN for 5 days significantly improved some measures of strength and muscle endurance compared to placebo. However, it is unclear whether these changes were primarily due to creatine and/or nitrate. Some improvement in exercise performance was also reported with acute [210] and 8-weeks [211] supplementation of a pre-workout supplement containing 2 g/day of CrN. However, since the supplement contained caffeine and other ergogenic nutrients, the benefits cannot be attributed to CrN. Further, it remains to be determined whether CrN supplementation has any additional benefit than simply co-ingesting CrM another source of nitrate (e.g., beet powder).

In terms of safety, since nitrates may lower blood pressure, there has been some concern that CrN may promote hypotension, particularly around intense exercise and/or if individuals take higher than recommended doses. Several studies have assessed safety of acute and chronic CrN supplementation. Dalton et al. [36] reported that ingestion of up to 6 g of CrN for 6 days does not negatively affect resting hemodynamics, response to a postural challenge, the ability to perform high-intensity exercise, or clinical chemistry profiles. Joy and colleagues [212] reported that 28 days of CrN supplementation (1 and 2 g/day) during training had no adverse effects on clinical blood chemistries compared to a non-supplemented group. Galvan and coworkers [29] also found no adverse effects after 28 days of supplementation (3 g/day). Finally, Jung and associates reported no adverse effects of participants consuming a pre-workout supplement containing 2 g/day of CrN for 8 weeks. Thus, CrN appears to be safe when taken in these amounts and timeframes. However, CrN has only been approved as a dietary supplement by the U.S. FDA at levels of 750 mg per day, which is below any meaningful level expected to increase muscular creatine levels and performance. Based on this analysis, there is some evidence showing

CrN may serve as a bioavailable and effective source of creatine. However, most studies have used higher than recommended doses, and those studies show that CrN is not more effective than CrM supplementation.

#### *9.6. Buffered Creatine Monohydrate*

In the early 2010s, a "buffered" or "pH-correct" form of CrM was heavily marketed as a more bioavailable source of creatine than CrM [213]. According to the patent [214], CrM was better stabilized by adding an alkaline powder (e.g., soda ash, magnesium glycerol phosphate, bicarbonate) to CrM (or other purported forms of creatine) in order to increase pH between 7–14. Consequently, they developed CrM that was "synthesized to a pH of 12" (CrM-Alk) and claimed that due to greater stability in preventing the conversion of CrM to creatinine, CrM-Alk was up to 10 times more bioavailable than CrM [213]. Therefore, 1.5 g of CrM-Alk was purported to be equivalent to ingesting 10–15 g of CrM. Additionally, the company theorized that since less CrM-Alk was needed to be ingested, there would be fewer side effects than CrM [213]. To support these claims, the manufacturers cite a non-peer reviewed report from Bulgaria on their website [215]. In this report, 24 healthy Olympic level soccer players were administered increasing doses of CrM-Alk or CrM at one-month intervals (i.e., 0, 1.5, 4.5, and 6 g/day). The authors reported that CrM-Alk promoted less of an increase in urine creatinine than CrM despite changes being <0.2% different between groups at each time point; that urine pH increased by 0.65 in the CrM-Alk group, but only 0.1 in the CrM group (CrM-Alk 5.27 to 5.92; CrM 5.5 to 5.6); and, peak oxygen uptake increased in the CrM-Alk group (<30 mL/min over time or <1.0% for a trained individual). No differences in body weight were reported. These investigators concluded the CrM-Alk group "outperformed creatine monohydrate as a creatine product" despite not performing any statistical analysis to determine if these minimal differences were statistically significant. Thus, the report does not validate claims than CrM-Alk supplementation is a more bioavailable, efficacious, or safer form of creatine than CrM.

Conversely, in a very well-controlled clinical trial, Jagim and colleagues [28] compared the effects of CrM-Alk supplementation at recommended and equivalent doses to CrM during 28 days of training in resistance-trained athletes with no recent history of creatine supplementation. In a double-blind manner, 36 resistance-trained participants were randomly assigned to ingest CrM (4 × 5 g/day for 7-days, 5 g/day for 21-days), CrM-Alk at recommended doses (1.5 g/day for 28-days), or CrM-Alk with equivalent doses to CrM (4 × 5 g/day for 7-days, 5 g/day for 21-days). Muscle biopsies, dual-energy x-ray absorptiometry (DXA) determined body composition, and performance measures were obtained after 0, 7, and 28 days of supplementation. Results revealed that neither recommended doses of CrM-Alk or loading and maintenance equivalent doses of CrM-Alk to CrM promoted greater changes in muscle creatine content, body composition, strength, or anaerobic capacity than CrM (see Figure 13). In fact, muscle creatine content was not significantly increased after 7 or 28 days of supplementation at recommended doses (−6.4 ± 37.8; 13.7 ± 42.2 %, respectively). There was some evidence that ingesting higher doses of CrM-Alk increased muscle creatine content after 28 days (6.2 ± 29.2; 27.3 ± 49.1%, respectively), but these values were less than observed with CrM (23.5 ± 49.0; 50.4 ± 44.8%, respectively). Thus, while high doses of CrM-Alk may increase muscle creatine content to some degree over time, there is no evidence that CrM-Alk is up to 10 times more bioavailable than CrM and/or recommended doses are efficacious. Additionally, was no evidence that CrM-Alk promoted greater training adaptations than those taking CrM or that participants taking CrM-Alk experienced fewer side effects than those taking CrM. Therefore, buffered creatine monohydrate is classified in some evidence to the support bioavailability, efficacy, and safety categories, but there is no evidence that buffered creatine is better.

**Figure 13.** Changes in muscle creatine content, fat-free mass, and 30 s cycling sprint performance after 7 and 28 days of CrM-Alk supplementation at 1.5 g/day recommended doses (), CrM-Alk supplementation of 20 g/day for 7 days and 5 g/day for 21 days (■), or CrM supplementation of 20 g/day for 7 days and 5 g/day for 21 days (•). Adapted from Jagim et al. [28].

Table 3 summarizes results of studies assessing creatine-containing compounds in which there is some evidence supporting bioavailability, efficacy, and/or safety. As described above, while some effects were observed compared to placebo ingestion and in some instances have comparable effects on performance as CrM, none of these forms have been shown to promote greater creatine retention in muscle than CrM.

**Table 3.** *Cont.*

R = randomized; DB = double-blind; *p* = placebo; SB = single blind; C = crossover; yrs = years; PLA = placebo, CHO = carbohydrate; PRO = protein; Cr = creatine; CrM = creatine monohydrate; CC = creatine citrate; CPY = creatine pyruvate; caPYR = calcium pyruvate, MgCr-C = magnesium creatine chelate, CEE = creatine ethyl ester; Cr-HCl = creatine hydrochloride; CrN = creatine nitrate; CNN = creatinine; CrM = Alk = buffered creatine; PCr = phosphocreatine; TCr = total creatine; AUC = area under the curve; FFM = Fat-Free Mass; BIA = bioelectrical impedance; GI = gastrointestinal; MRS = magnetic resonance spectroscopy; DW = dry weight.

#### **10. No Evidence to Support Bioavailability, Efficacy, and Safety**

#### *10.1. Other Creatine Salts*

While there is some bioavailability, efficacy, and safety data on CC and CPY, little to no data are available on several other creatine salts listed in Table 1. A patent from Negrisoli and Del Corona [31] disclosed several hydrosoluble organic salts of creatine including creatine maleate, creatine fumarate, creatine tartrate, and creatine malate. The inventors claimed that these creatine salts had solubilities of 10, 19, 3, 8.5, and 4.5 g/100 mL (%), respectively. Since salts are relatively weak bonds, it is likely that these creatine salts would increase creatine in the blood at equimolar doses that are generally about 1.3–1.6 times greater than CrM. However, there are no data indicating that these creatine salts increase blood creatine content, increase tissue creatine content, have any ergogenic value, are safe for long-term supplementation, or are more effective sources of creatine than CrM. A patent disclosing creatine ascorbate was also filed in the late 1990s [33]. The rationale was to provide a means of increasing creatine and ascorbic acid availability to improve exercise capacity while supporting the immune system [33]. However, we are not aware of any pharmacokinetic or exercise related studies to test this hypothesis and benefits would seemingly be similar and more effectively dosed by co-ingestion of CrM and vitamin C. Finally, there has been interest in tri-creatine orotate (CO) as a creatine salt (71% creatine by molecular weight) and a few raw material suppliers offer CO as a source of creatine to manufacturers [30,176,177]. Supplement companies who sell creatine orotate claim it provides creatine and orotic acid that is purported to aid in the production of carnosine in the muscle, and therefore improves muscle-buffering capacity. However, as of this writing, we are aware of no data showing bioavailability and/or efficacy of CO supplementation [216]. The European Food and Safety Authority has also expressed concerns about the potential cancerogenic effects of orotic acid [176,177]. Therefore, CO does not seem to be a good alternative for CrM in dietary supplements particularly when co-ingestion of effective doses of CrM and beta alanine would seeming be more effective. Based on this analysis, these creatine salts are classified in the no evidence to support bioavailability, efficacy, and safety category. Therefore, they cannot be considered more effective than CrM.

#### *10.2. Creatine Serum*

As noted above, there has been interest in developing shelf-life stable liquid, gels, and/or beverages containing creatine. The theoretical rationale has been that these types of products may be more convenient to consume, absorbed faster into the blood, and/or promote a greater efficiency in transport of creatine to the muscle. One product that was heavily marked in the late 1990s and early 2000s is "creatine serum" (CS). This product claimed to deliver 2.5 g of creatine per 5 mL oral dose by providing a "creatine phosphate complex" that was designed to be absorbed via mucosal thereby bypassing the supposed degradation of creatine to creatinine through digestion [217]. Their rationale was based on general pharmacokinetic absorption studies indicating that drugs and/or nutrients are absorbed faster through the mucosal lining in the mouth. However, when researchers evaluated the creatine content of CS, they found that CS contained <10 mg of creatine and 69 mg of creatinine per 5 mL dose in multiple samples and lot numbers [38]. Additionally, they found that one 5 mL oral dose of CS purportedly providing 2.5 g of creatine had no effect on plasma creatine levels (same as water) whereas ingestion of 2.5 g of CrM increased plasma creatine levels to about 300 μmol/L after one hour of ingestion and declining in a classical manner throughout the next 8 h (see Figure 14A) [38]. No changes in creatinine levels were seen among participants ingesting CS, CrM or water. Consequently, this study shows that CS does not contain creatine and has no bioavailability in the blood [38]. To further assess the bioavailability of serum creatine, Kreider and colleagues [26] evaluated the effects of ingesting 5 mL of a flavored placebo; 5 mL of CS (purportedly providing 2.5 g of CrM); 8 × 5 mL doses of CS per day (purportedly providing 20 g/day of CrM); and 4 × 5 g doses of CrM (20 g/day) for 5 days on muscle creatine, phosphocreatine and

content. Results revealed that CrM loading significantly increased total muscle creatine (+31%) and phosphocreatine (+16%) (see Figure 13). However, CS ingestion at recommended and equivalent doses had no effects on muscle free creatine, phosphocreatine, total creatine content, or ATP concentrations. Collectively, these studies show that CS is not a bioavailable source of creatine, and therefore can have no creatine-related efficacy. Therefore, it is classified in the no evidence to support category, and there is no evidence that CS outperforms CrM. Unfortunately, this product remains in the marketplace despite data showing it is a completely ineffective source of creatine.

**Figure 14.** Changes in plasma creatine levels after oral ingestion of water, 5 mL of creatine serum (CS) purportedly providing 2.5 g of CrM, and 2.5 g of CrM in solution (**A**) [26] and 5 days of 5 mL of CS purportedly providing 2.5 g of creatine (CS-LD), 5 mL of a flavored placebo (PL-LD), 8 × 5 mL of CS (CS-HD) purportedly providing 20 g/day of creatine, 8 × 5 mL of flavored placebo (PL-HD), or 4 × 5 g/day of CrM (**B**) [26], Adapted from Harris et al. [38] and Kreider et al. [26].

#### *10.3. Creatyl-L-Leucine*

Creatyl-L-Leucine (CLL) has been marketed as "super creatine" [218]. As described in a patent [219], CLL is claimed to be "stable aqueous composition" of an "amide-protected, biologically-active form of creatine (creatyl-amide) molecule" that is "stable across a wide range pH's and temperatures" and "can provide a wide range of physiological benefits including, for example, regeneration of ADP to ATP in muscle tissue, increasing the serum concentration of creatine, increasing muscle fiber size/cross-sectional area and lean body mass, activating satellite cells, enhancing memory and cognitive function, enhancing the functional capacity of a mammal having a neuromuscular disease, increasing muscular strength, endurance and/or power, enhancing cognitive function in infants with inborn errors of creatine metabolism, and/or alleviating the deleterious effects of sleep deprivation". Analysis of the structure of CLL (see Figure 15) indicates that CLL does not contain a creatine molecule. Additionally, amide bonds are generally very strong, so pharmacokinetic data would need to show that CLL breaks down into creatine, increases creatine in the blood, and increases tissue creatine content to establish that CLL is a bioavailable source of creatine.

**Figure 15.** Comparison of creatine, creatine monohydrate, L-leucine, and creatyl-L-leucine chemical structures.

As of this writing, only two published articles have assessed the safety and/or efficacy of CLL. The first study was toxicology assessment of the administration of large doses of CLL in rats [220]. This study found that CLL did not cause mortality, toxic effects, or adverse effects in rats administered CLL for 90-days by oral gavage at doses of 1250, 2500, and 5000 mg/kg/day. More recently, da Silva [221] conducted an elegant study assessing the effects of feeding 24 rats either a control diet, a diet containing 4.0 g/kg/day of CrM, or a diet containing 6.56 g/kg/day of CLL for 7 days on arterial delivery of creatine, tissue uptake, and storage. According to the researchers, for a 70 kg individual, this would equate to a dose of 17.6 g/day of CrM and 28.9 g/day of CLL providing equimolar amounts of creatine if CLL based on the molecular weight of creatine if CLL degraded into creatine. As shown in Figure 16, rats fed CrM experienced significant increases in creatine concentrations in arterial plasma (+7-fold), portal vein plasma (+10-fold), muscle creatine content (+1.63 fold from control, and +1.53-fold from CLL) while tending to increase brain creatine content (*p* = 0.052) compared to controls. These changes were significantly greater than rats fed a control or CLL containing diet.  The researcher concluded that provision of large doses of CLL to rats did not increase creatine bioaccumulation indicating that CLL is poorly absorbed by the intestine and is not a bioavailable source of creatine.

**Figure 16.** Blood, muscle, and brain creatine content in response to rats fed a control diet, 4.0 g/kg/day of creatine monohydrate (CrM), or 6.56 g/kg/day of creatyl-L-Leucine (CLL) for 7 days. (**A**) presents arterial plasma creatine concentration, (**B**) presents portal vein creatine concentration, (**C**) presents muscle creatine content, and (**D**) presents brain creatine content data for each group. Data are means ± standard deviations. \*\*\*\* = *p* < 0.0001, \*\*\* = *p* < 0.001, \*\* = *p* < 0.01, \* *p* < 0.05, ns = not statistically significant between groups identified in brackets. Adapted from da Silva [221].

Several other studies have been recently conducted on CLL supplementation with human participants by experienced researchers at respected institutions with reports of results submitted in ongoing lawsuits [222–226]. These reports provide additional data showing CLL is not degraded into creatine upon oral ingestion [222,223,226], CLL does not increase blood creatine content [222,223,226], and CLL does not increase muscle [224,225] or brain creatine content [224] even when administered at doses much higher than found in marketed products containing CLL in humans [222–226]. Additionally, several of these studies reported that ingesting equivalent doses of CrM promoted significantly greater increases in blood [222,223,226] and tissue creatine content [225] than those ingesting CLL and CLL ingestion was no different than placebo controls [225,226]. Thus, available evidence indicates that CLL is not creatine, CLL is not a bioavailable source of creatine, and CLL is not "super creatine" compared to CrM. Therefore, CLL is listed in the no evidence category.

#### *10.4. Creatinol-O-Phosphate*

Figure 17 shows the chemical structure of creatinol-O-phosphate (COP). Creatinol in the form of COP is not creatine, nor was it intended to increase muscle creatine content. Rather, it was initially studied in the 1970s to intravenously deliver phosphate to improve myocardial function and reduce arrhythmias during ischemic conditions [227–230]. For example, Melloni and colleagues [228] investigated the effects of intravenous administration of 1020 mg, 2040 mg, and 3060 mg of COP compared to placebo on arterial blood pressure, heart rate and arrhythmias. The researchers found that COP administration increased blood phosphate levels as well as urinary excretion of phosphate and creatinine. Phosphate loading has been found to increase myocardial ejection fraction during exercise and maximal aerobic capacity [231,232] and is considered an ergogenic aid for endurance athletes [14]. While this is unrelated to creatine supplementation, the increase in urinary creatinine excretion led some to speculate that creatinol may act as a precursor of creatine and thereby serve as a source of creatine the body [228]. However, pharmacokinetic studies indicated that absorption of COP was complete when administered intramuscularly and distributed primarily to the kidney, liver, and heart and that COP could cross myocardial cell membranes [230]. One study from 1975 has been reported by others to show that intramuscular and intravenous administration of COP increased handgrip performance [233]. However, it is difficult to find details about this study. We are also not aware of any study that has evaluated whether oral COP has any effect on muscle creatine levels or exercise performance. Nevertheless, some companies have included COP as a source of creatine in dietary supplements and energy drink beverages. There is no evidence that oral COP ingestion has any effect on muscle creatine content or creatine-related metabolism. Claims that oral COP is a source of creatine and/or is more bioavailable than CrM are not supported.

**Figure 17.** Chemical structure of Creatinol-O-Phosphate.

Table 4 summarizes the results of studies that have evaluated sources of creatine that currently have no evidence supporting of bioavailability, efficacy, and/or safety. As can be seen, there are limited published data on these purported sources of creatine, and the available evidence indicates that they are not bioavailable sources of creatine.

**Table 4.** Creatine containing compounds that have no evidence of bioavailability, efficacy, and/or safety.

R = randomized, *p* = placebo, DB = double-blind, SB = single blind; C = crossover, PLA = placebo, Cr = creatine; TCr = total creatine; CrM = creatine monohydrate, CS = creatine serum; CLL = creatyl-L-leucine; COP = creatinol-O-phosphate; ↑ = increase.

#### **11. Regulatory Status**

#### *11.1. United States*

In the United States (US), congress enacted the Dietary Supplement Health and Education Act (DSHEA) of 1994 that placed dietary supplements in a special category of foods under the jurisdiction of the FDA. According to DSHEA, a dietary supplement is a product intended to supplement the diet, ingested orally, and contains a "dietary ingredient". Dietary ingredients include vitamins, minerals, amino acids, herbs, botanicals, and other substances such as extracts, metabolites, or concentrates of those substances [14,234]. Dietary supplements can be delivered in powders, pills, capsules, hard and chewable tablets, soft gels, gummies, liquids, and even properly labeled energy bars that are intended for oral ingestion. However, they cannot include products promoted for sublingual, intranasal, transdermal, injected, or in any other route of administration [14]. DSHEA also established laws for FDA oversight over "new dietary ingredients" (NDI), which are ingredients that introduced to the marketplace after DSHEA was enacted [235]. A dietary supplement containing an NDI is deemed adulterated by the FDA, and therefore may not be lawfully distributed, unless (1) the NDI has "been present in the food supply as an article used for food in a form in which the food has not been chemically altered" or (2) there is a "history of use or other evidence of safety" that is submitted to the FDA for at least 75 days before selling the product (i.e., an "NDI Notification"). Dietary ingredients that were sold in the U.S. prior to October 15, 1994, were considered "grandfathered," and therefore not NDIs subject to these requirements. This included CrM since it was introduced into the U.S. market in 1993 [236].

Since all of the other purported sources of creatine described above were introduced into the U.S. marketplace after 15 October 1994, they are considered NDI's and manufacturers and distributors were expected to notify the FDA about these ingredients (See Section 413(d) of the Federal Food, Drug, and Cosmetic Act (the FD&C Act), 21 U.S.C. 350b(d)) [235] unless they meet the "present in the food supply" exemption noted above. An NDI Notification should include documentation of how the product containing the NDI is "reasonably expected to be safe" along with (1) the name of the new dietary ingredient (or Latin binomial name if it is an herb or botanical); and (2) "a description of the dietary supplement that contains the new dietary ingredient, including (a) the level of the new dietary ingredient in the product, (b) conditions of use of the product stated in the labeling, or if no conditions of use are stated, the ordinary conditions of use, and (c) a history of use or other evidence of safety establishing that the dietary ingredient, when used under the conditions recommended or suggested in the labeling of the dietary supplement, is reasonably expected to be safe" [14]. Once submitted, the FDA has 75 days to object to the notification. If the FDA does not respond within this timeframe, the NDI can be included in dietary supplements and legally sold in the U.S. market. However, it is important to understand that an NDI Notification only indicates that the FDA considers the NDI to be reasonably be considered as safe for human consumption. It does not affirm efficacy and/or validate any claims made about the NDI.

Since DSHEA and FDA regulations do not provide sufficient clarification on many issues, there has been a lot of confusion in the dietary supplement industry on what is an NDI, what manufacturing or other changes made to an ingredient cause it to be a "new" ingredient, when an NDI Notification is required, and what information it should contain. To provide clarification, the FDA released a "Draft Guidance for Industry" entitled "Dietary Supplements: New Dietary Ingredient Notifications and Related Issues" in July of 2011. However, that draft guidance prompted even more confusion and controversy, so the FDA released a revised draft guidance in 2016. While a guidance does not carry enforcement authority like a law or regulation, it provides the FDA's perspective of how they interpret the laws and regulations related to NDI's to help dietary supplement manufacturers know whether they are required to submit an NDI Notification to FDA, how to prepare NDI Notifications consistent with FDA review expectations, and how to improve the quality of submissions [235]. The 2016 Draft Guidance has also been criticized for a lack

of clarity concerning what was considered a grandfathered ingredient and whether an NDI Notification was required if another manufacturer had already submitted an NDI Notification among other issues. This led to ingredients that should have been considered to be NDIs entering the marketplace with a notification, and several NDI Notifications being rejected by the FDA for lack of adequate safety data and/or other issues.

As an alternative to submitting an NDI Notification, some companies have pursued obtaining "Self-Affirmed" "Generally Recognized as Safe" (GRAS) status by conducting toxicology studies on animals and having scientific experts review the safety data and affirm the ingredient was reasonably expected to be safe. In this case, the company commissions studies to assess safety (e.g., toxicology studies in animals) and maintains internal documents to show safety if requested by the FDA. Once an ingredient is self-affirmed as GRAS, it can be introduced into the food supply, and is then not required to submit an NDI Notification to FDA to be included in dietary supplement under the "present in the food supply" exemption noted above. Companies can voluntarily submit their ingredient's GRAS determination to FDA, however, there is no requirement to do so, and therefore a company's GRAS self-affirmation can remain private. While lawful, the FDA has expressed some concern about this approach and discourages dietary supplement manufacturers from self-affirming GRAS to avoid submitting an NDI Notification.

After a dietary supplement is entered into the U.S. market, the FDA can restrict or ban its sale if it is deemed adulterated (e.g., unsafe). The FDA works with the Federal Trade Commission (FTC), who has jurisdiction over marketing claims that companies make about dietary supplements in advertising to regulate the dietary supplement industry. The FTC can act against companies for disseminating false, misleading, or unsubstantiated claims about dietary supplements. Marketing claims made for dietary supplements can also be challenged in litigation brought by consumers or competitors. This background provides the basis for understanding how various purported forms of creatine have entered the US market. The legal and regulatory status of CrM in dietary supplements is indisputable because it appeared on the US market in 1993 [236], and there was a large body of evidence showing it was reasonably expected to be safe. Since then, AlzChem Trostberg GmbH (the Germany manufacturer of CrM) voluntary submitted a GRAS application to the FDA that was not acted upon, meaning it could claim that CrM has FDA approved GRAS status [237,238]. Consequently, CrM is considered GRAS for inclusion as a dietary ingredient in dietary supplements, energy drinks, protein bars and powders, milkshakes, meal replacement powders and bars, meat replacement products, powdered drink mixes, and functional foods. As of this writing, CrM is the only form of creatine that is listed on the FDA's inventory of GRAS notices [239] (see Table 5). As noted above, a company is not required to submit its GRAS self-affirmation to FDA, and therefore it may not be public information.

**Table 5.** Regulatory status of nutrients marketed as creatine supplements in the United States of America.

FDA = Food and Drug Administration. \* Data retrieved from AHPA NDI Database http://ndi.ahpa.org/ (accessed on 1 January 2022).

The legal status of other purported sources of creatine is less clear. According to the FDA's NDI Notification database, since 1995, NDI Notifications for creatine pyruvate (1998), creatine ethyl ester (2003), creatine ethyl ester HCL (2004), tri-creatine orotate (2003), β-creatine (2010), creatine nitrate (2011 and 2017), and creatine acesulfame (2018) have been submitted. Several of these notifications were initially objected to by the FDA citing: (1) the form of creatine may not be a legal dietary ingredient as defined by the FD&C Act §201(ff); (2) inadequate safety information to conclude that the form of creatine is reasonably expected to be safe; and/or (3) a lack of information about the chemical identity of the creatine form [25]. However, creatine pyruvate at doses of 5–10 g/day and creatine nitrate at 750 mg per day have not been objected to by the FDA and can be sold as dietary ingredients. The AHPA NDIN database does not include notifications for creatine maleate, creatine fumarate, creatine tartrate, creatine ascorbate, creatine citrate, magnesium creatine chelate, creatine HCL, alkaline creatine (although it is a buffered form of CrM), creatine serum, CLL, or COP. Since some of these sources of creatine are ingredients in dietary supplements, any company selling these nutrients would have to have documentation that the ingredient was on the market in the US before 15 October 1994 or is present in the food supply in a form that is not chemically altered. While FDA GRAS notifications are published and are accessible to the public, self-affirmed GRAS files are not published, which makes them difficult to search and to validate its content. Based on a press release, we identified one self-affirmed GRAS affirmation for a creatine chelated with Mg (Creatine MagnaPower®).

#### *11.2. International Regulation*

Every country has independent laws and regulations governing dietary supplements. Some follow FDA guidance while others classify dietary supplements and drugs and have additional oversight, approval, and/or limitations about dosages [240]. Creatine monohydrate can be legally sold throughout the world although some countries limit the amount to of CrM that can be included per dose (e.g., no more than 3–5 g/serving). Some of the other forms of creatine marketed as ingredients for dietary supplements are not permitted to be included in dietary supplements in their country due to a lack of safety data. Therefore, except for CrM, one cannot assume that of the purported other sources of creatine described above can be legally sold as a dietary supplement in all countries. The following provides a brief overview in major markets that creatine is sold and regulatory oversight. Table 6 describes the responsible agencies and regulatory status of creatine containing dietary supplements in various countries. As can be seen, CrM remains the only source of creatine that is approved for sale in Australia, Canada, China, the European Union, Japan, and South Korea. Additionally, it is the only source of creatine that has approved health claims in the European Union, Canada, Japan, and South Korea.

**Table 6.** Regulatory status of nutrients marketed as creatine supplements primary international markets.

CrM = creatine monohydrate.

#### *11.3. Assessment and Guidance for Industry*

The regulatory status of CrM is unequivocal in the global markets as a dietary or food supplement [25]. However, the scientific basis and regulatory status of other forms of creatine continues to be less clear. Since our last review in 2011, more data is available about several forms of creatine and several now have data providing some support as to efficacy and safety while others do not. No alternative form of creatine has shown superior bioavailability, efficacy or safety compared to CrM. Consequently, despite marketing hyperbole, CrM remains the gold standard to compare other forms of creatine with the strongest bioavailability, efficacy, and safety portfolios. Alternatives to CrM continue to be prevalent in the marketplace, including several that do not appear to meet regulatory requirements in several countries.

In the US, a major factor in determining whether an NDI notification is required to be submitted to the FDA is whether the NDI has been present in the food supply and/or has been chemically altered from its original form [26]. Any change in the chemical structure of an ingredient is assumed to alter the biological activity of the ingredient thereby requiring toxicology studies to establish that the NDI is reasonably expected to be safe as altered and that the biological behavior is comparable to the native ingredient. Most of the forms of creatine listed in Table 1 have been chemically altered in some way (e.g., covalently binding or complexing) to the creatine molecule. Some have clearly rearranged the creatine molecule. Therefore, they should have all been submitted as and NDI notification to the FDA prior to marketing. Yet, as described above, only nine of these newer forms are listed in the IND notification inventory. Of these, the FDA initially rejected some of the IND notification applications, yet the forms were sold for years before finally submitting an acceptable IND notification application or Self-Affirming GRAS. Even then, many have little to no data supporting bioavailability and/or efficacy despite making bold claims that the source is more bioavailable, effective, and/or a safer form of creatine than CrM.

The reason why alternate marketed forms of creatine are in the marketplace without pre-market IND notification is likely due to confusion over legal definitions of dietary supplements, natural health products, and/or food additives in different countries as well as what is meant by chemical alteration in a nutrient. Regardless, confusion of over laws regulating dietary supplements combined with inadequate enforcement by regulators has created an environment where there are often little consequences of non-compliance. For example, studies published in 2003 clearly showed that creatine serum was not a bioavailable source of creatine, yet it continues to be sold as a creatine-containing product. While alternative forms of creatine are unlikely to pose a health risk, they are typically more expensive than CrM. Additionally, misleading claims that lower doses of an alternate form of creatine are as effective as CrM may limit the benefits consumers may achieve from creatine supplementation. Given the health benefits of creatine, availability of ineffective sources of creatine or recommendations to take less creatine than needed to increase creatine stores in the muscle and/or brain can limit the benefits theses populations may derive from creatine supplementation [5]. Thus, we give the following recommendations as guidance to researchers and industry as they consider developing new dietary ingredients containing creatine.

#### **12. Summary**

CrM supplementation increases muscle phosphagen levels, improves repetitive highintensity exercise performance, and promotes greater training adaptations [15]. No significant side effects other than weight gain have been reported from CrM supplementation despite widespread use throughout the world. Research on CrM has served as the basis to establish professional guidelines, recommendations, and establish regulation. CrM remains the only source of creatine that has substantial evidence of bioavailability, efficacy and safety and is considered GRAS by the U.S. FDA, is approved for use with accompanying health claims in the EU, has been extensively reviewed and approved by Health Canada, and is approved to be sold in major global markets. The bioavailability, efficacy, safety, and regulatory status of other purported sources of creatine are less clear, with only a few having some data supporting efficacy compared to placebo (see Table 7). However, there is no evidence that other "forms" of creatine are more bioavailable, effective, or safer forms of creatine compared to CrM. We recommend that companies interested in developing and marketing novel forms of creatine ensure the purported source contains the creatine molecule and conduct high-dose safety data in animals, pharmacokinetic studies to show the source of creatine increases blood and tissue concentrations of creatine, and comparative effectiveness studies to support structure and function claims. Additionally, the should company clearly list the amount of creatine contained in the supplement on supplement facts labels so consumers can make an informed decision about whether that purported source of creatine may deliver enough creatine to increase tissue creatine content by physiological levels needed to effect exercise and/or health.

**Table 7.** Categorization of purported sources of creatine based on bioavailability, efficacy, and safety.

**Author Contributions:** Conceptualization, R.B.K., R.J. and M.P.; writing—original draft preparation, R.B.K.; writing—review and editing, R.J. and M.P.; funding acquisition, R.B.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC of selected papers of this Special Issue are being funded by Alzchem, LLC. (Trostberg, Germany), which manufactures creatine monohydrate. The funders had no role in the writing of the manuscript, interpretation of the literature, or in the decision to publish the results.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to thank all the study participants, scholars, and funding agencies who have contributed to the research cited in this manuscript. We would also like to thank Ivan Wasserman (U.S.), Toshitada Inoue (Japan/South Korea), and John Doherty (Canada) for reviewing the local regulations.

**Conflicts of Interest:** R.J. and M.P. are researchers and principals for Increnovo, LLC, which conducts research, develops intellectual property, and consults with industry about raw ingredients and product formulations. They have filed patents for creatine while being employed by SKW Trostberg/Degussa AG (now Alzchem), from 1999 to 2007, with all the patents being expired or abandoned (WO2006015774A1, US20110123654A1, WO2003071884A1, US20050096392A1, WO2006122809A1, WO2002052957A1, WO2003047367A1, US20040006139A1, US20020072541A1, DE10244281A1, DE10119608A1), and conducted research on various forms of creatine. R.B.K. has

conducted sponsored research on dietary supplements including creatine through grants awarded to the universities he has been affiliated with, received honorarium for presenting research related to dietary supplements and creatine at industry-sponsored scientific conferences, has served as an expert witness on cases related to dietary supplements, including past and current cases related to creatine, and is acting Chair of the Scientific Advisory Board on Creatine for Alzchem. He has also presented research related to creatine at a number of international conferences.

#### **References**

### *Review* **Timing of Creatine Supplementation around Exercise: A Real Concern?**

**Felipe Ribeiro 1,2, Igor Longobardi 1, Pedro Perim 1, Breno Duarte 1, Pedro Ferreira 1,2, Bruno Gualano 1,3, Hamilton Roschel <sup>1</sup> and Bryan Saunders 1,4,\***

**Abstract:** Creatine has been considered an effective ergogenic aid for several decades; it can help athletes engaged in a variety of sports and obtain performance gains. Creatine supplementation increases muscle creatine stores; several factors have been identified that may modify the intramuscular increase and subsequent performance benefits, including baseline muscle Cr content, type II muscle fibre content and size, habitual dietary intake of Cr, aging, and exercise. Timing of creatine supplementation in relation to exercise has recently been proposed as an important consideration to optimise muscle loading and performance gains, although current consensus is lacking regarding the ideal ingestion time. Research has shifted towards comparing creatine supplementation strategies pre-, during-, or post-exercise. Emerging evidence suggests greater benefits when creatine is consumed after exercise compared to pre-exercise, although methodological limitations currently preclude solid conclusions. Furthermore, physiological and mechanistic data are lacking, in regard to claims that the timing of creatine supplementation around exercise moderates gains in muscle creatine and exercise performance. This review discusses novel scientific evidence on the timing of creatine intake, the possible mechanisms that may be involved, and whether the timing of creatine supplementation around exercise is truly a real concern.

**Keywords:** dietary supplements; ergogenic aid; hypertrophy; resistance training; sports nutrition; strength; supplementation

#### **1. Introduction**

Athletes (and physically active individuals) are interested in nutritional strategies that are aimed at enhancing exercise performance. Creatine (Cr) deserves a special place among the plethora of ergogenic supplements, as it is one of the most studied and scientifically supported supplements on the market [1,2]. Creatine is a naturally occurring non-protein nitrogen compound synthesised in the liver and kidney from precursor amino acids, arginine, glycine, and methionine. Most of the body's Cr is found in muscle (95%), of which two-thirds are stored as phosphorylcreatine (PCr), the remaining third as free Cr [3], with less than 5% found in other tissues, such as the brain and testes [4]. In a seminal study by Harris et al. (1992), it was demonstrated for the first time in humans that Cr supplementation, at varying doses of 20–30 g/day, ingested over several individual 5 g doses throughout the day, could increase total intramuscular Cr content (TCr = PCr + Cr) by as much as 20% [5]. Numerous subsequent studies have shown the efficacy of Cr supplementation in increasing muscle Cr content, including using more gradual loading protocols [6–8].

**Citation:** Ribeiro, F.; Longobardi, I.; Perim, P.; Duarte, B.; Ferreira, P.; Gualano, B.; Roschel, H.; Saunders, B. Timing of Creatine Supplementation around Exercise: A Real Concern? *Nutrients* **2021**, *13*, 2844. https://doi.org/10.3390/nu13082844

Academic Editor: Ajmol Ali

Received: 26 June 2021 Accepted: 10 August 2021 Published: 19 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Several factors could influence the individual intramuscular increase in TCr and subsequent performance benefits as a consequence of Cr supplementation, including baseline muscle Cr content, type II muscle fibre content and size, and habitual dietary intake of Cr and aging [9,10]. Interestingly, it has been known for some time that exercise can enhance Cr loading in muscle [5], and the specific timing of Cr supplementation in relation to exercise has more recently been touted as an important consideration, in order to optimise training gains, although the current consensus on its importance is lacking. Emerging evidence suggests that post-exercise Cr ingestion may provide superior benefits compared to pre-exercise consumption [11,12], although several methodological limitations presented by these investigations currently preclude definitive interpretations of these results.

The purpose of this narrative review is to summarise and discuss current evidence and new emerging questions on the influence of Cr supplementation timing, in regard to exercise, on muscle Cr content and physical performance.

#### **2. Creatine Supplementation**

Studies show that creatine supplementation in doses of 5–20 g/day for >5 days can increase intramuscular Cr and PCr to the point of saturation [8,13]. This increase in PCr is associated with the main mechanism of action, regarding the ergogenic effect of Cr supplementation [14]. Phosphorylcreatine can provide an inorganic phosphate (Pi) molecule for the resynthesis of ATP via the Cr kinase reaction, in which Pi donation from PCr degradation is used by adenosine diphosphate (ADP) and, consequently, increases ATP resynthesis. Creatine phosphokinase is the enzyme that catalyses this reaction and is limited only by the concentration of its substrates and products, namely Cr and PCr [15]. This phosphagen system, also termed the ATP-CP system, is the fastest way to supply ATP for skeletal muscle metabolism [16]. Precisely for this reason, the ATP-CP system is related to high-intensity and short-duration exercises [17] and, thus, is associated with greater total work capacity [1]. In this respect, the ATP-CP system serves as an important regulator of muscle metabolism, which explains the ergogenic benefits of Cr supplementation throughout training. Enhancing the capacity of ATP resynthesis should increase available energy during exercise, prolonging the work capacity of the skeletal muscles, delaying the onset of muscle fatigue, and improving performance.

Strong scientific evidence suggests that Cr can lead to beneficial improvements in exercise performance; however, there also appears to be some variations in the response to Cr supplementation due to a number of factors, which will be presented in the next section.

#### *Factors Modifying the Effect of Creatine Supplementation on Muscle Creatine Content*

Several factors have been shown to modify the effects of Cr supplementation on muscle Cr content. Daily dose and duration play important roles in how quickly and how much Cr stores are increased. Five to seven days of supplementation with a daily dose of 20 g·day−<sup>1</sup> is sufficient to saturate muscle creatine stores [5], which is approximately 140 to 160 mmol·kg−<sup>1</sup> of dry muscle. This has become a commonly employed dose in the literature and termed the "loading phase". Nonetheless, a more gradual dosing strategy of 3 g·day−<sup>1</sup> leads to similar increases, but over a longer period (~28 days; [8]). Greater increases in muscle Cr are shown in those with lower initial muscle Cr content [5], while carbohydrate co-supplementation may increase Cr uptake via insulin-mediated stimulation [6,18] of the Cr transporter, CreaT. Although this mechanism of insulin stimulated Cr uptake remains to be mechanistically confirmed, if it holds true, this will only be relevant within the first few days at high doses (e.g., days 1 to 3 at 20 g·day−1) of supplementation prior to saturation of muscle Cr stores [19], but may be more relevant at lower doses (e.g., 3–5 g·day<sup>−</sup>1, which takes up to 28 days to saturate). Indeed, the upper threshold of saturation across individuals appears remarkably consistent [1], meaning the dosing protocol will be important.

Exercise has also been shown to enhance Cr accrual in muscles. In the seminal study from Harris et al. (1992), various doses of Cr were given to healthy participants aged 20 to

62 years, with varying levels of fitness. An additional aim of this study was to determine the effect of exercise upon Cr uptake into muscles using a unilateral leg exercise model. Throughout supplementation, participants performed 1 h of cycling exercises in one leg, while the other leg rested. Results showed that exercise potentiated the resultant increase in intramuscular Cr, with greater increases in the exercised versus non-exercised leg. These were the first data to suggest that exercise may influence the Cr loading of muscles with supplementation. These data were subsequently supported by a further study that showed a 68% greater increase in total creatine content following supplementation when a singleleg exercise (cycling at 60–70% of maximal heart rate until exhaustion) was performed [20] (Robinson et al., 1999). Thus, exercise appears to enhance the accrual of intracellular TCr with Cr supplementation, although, again, the importance thereof will likely be linked to the daily dose employed. Since high doses (20 g·day−1) lead to saturation in as little as 5 days, it seems unlikely that quicker loading will have much impact. However, should supplementation occur more gradually, as with doses of 3–5 g·day−<sup>1</sup> over 28 days, then faster loading might incur earlier and greater benefits. It must be acknowledged that it is unclear if these studies showed an increased uptake of Cr into muscle, or an increased Cr retention. Logic would suggest that it is likely reflective of an increased muscle uptake although this should be mechanistically confirmed.

The influence of exercise on Cr loading is apparent; however, more recent investigations have suggested that the timing of Cr supplementation in relation to the exercise bout may be important, too [11,12,21,22]. To better understand why timing of supplementation in relation to exercise might be important, it is important to appreciate how exercise might enhance Cr uptake, which will be discussed in the following section.

#### **3. How Creatine Timing around Exercise May Influence Subsequent Loading**

#### *3.1. Creatine Concentration in the Bloodstream and Training Duration*

The mechanisms via which exercise may increase Cr uptake into muscles are not entirely understood and are hypothetical, as no study has experimentally demonstrated the mechanism behind this phenomenon. Nonetheless, one proposed mechanism via which timing of Cr ingestion in relation to exercise may modify the efficacy of supplementation is through exercise hyperaemia, namely increased blood perfusion to the working muscle (Figure 1A). Blood flow increases within one second of the onset of muscular contraction, and exercise can increase skeletal muscle blood flow by 100-fold compared to values seen at rest [23]; it is important to maintain adequate oxygen and nutrient delivery, in order to support the energetic demands of the skeletal muscles during exercise. The extent to which blood flow increases during and after exercise is influenced by factors such as the duration, type, intensity, and volume of exercise. This is important to note because muscle blood flow is closely matched to the metabolic demands of contractions induced by the exercise [24]. Theoretically, greater blood flow to the muscle could lead to greater delivery of Cr and, thus, enhance its uptake and retention, although this would primarily be restricted to the exercised muscles. An increase in blood flow kinetics as well as Cr transport to exercised muscles can result in greater delivery, retention, and metabolization of the nutrients to the exercised muscles [25]. Thus, if supplementation is provided around exercise, then circulating Cr could coincide with increased blood flow to the muscle (Figure 1B).

An important factor to consider, in regard to the timing of Cr supplementation in relation to exercise, is the time it takes for the Cr concentration to become elevated in the bloodstream. This is relevant to determine if ingestion of Cr pre- or post-exercise would provide distinct elevations of intramuscular Cr. In humans, Cr is actively absorbed from the gastrointestinal tract before entering the bloodstream to be delivered to various tissues throughout the body [26]. Creatine monohydrate absorption is close to 100% [27], and when 2 g of Cr is consumed in an aqueous solution, it reaches peak plasma concentration in approximately 1 h. This is similar to other protocols in which maximum plasma concentration of Cr occurred in <2 h when the dose administered was <10 g [18,28,29]. Although higher doses >10 g can take up to 2.5 to reach peak concentration in the blood [30], the

most employed single-dose of 5 g should peak around 1–2 h following ingestion, remaining elevated for a further4h[5,30]. This information could be crucial when optimizing supplement timing; if Cr-uptake is maximised when there is increased muscle blood flow, then individuals should look to coincide peak circulating Cr levels with hyperaemia.

**Figure 1.** The hypothetical mechanisms behind an exercise-mediated increase in total creatine content with creatine supplementation. (**Panel A**): exercise hyperaemia increases tissue perfusion, enhancing creatine delivery. Additionally, increased Na+/K+ pump activity during exercise supports the [Na+] gradient favouring creatine uptake by CreaT. Together, these effects may acutely potentiate the uptake and increase in total muscle creatine content. (**Panel B**): theoretical overlap of events according to the timing of creatine supplementation, in relation to exercise and its potential benefits regarding the delivery and uptake of creatine to the muscle. Created with BioRender.com.

> As an example, the duration of a typical resistance training session varies between 40 and 90 min, and elite bodybuilders reported an average of 60–70 min per training session [31]. Thus, should an individual supplement immediately pre-exercise or even during exercise, Cr would begin to accumulate throughout the training session, and it is possible that peak Cr concentration in the blood would still occur during exercise. Due to the exercise undertaken, this would lead to increased blood flow to the working muscles, which may lead to increased delivery and subsequent uptake of Cr, explaining, at least partially, the greater increases in Cr loading shown previously [5,30]. This also suggests that pre-exercise Cr supplementation may be more effective at increasing muscle Cr content than post-exercise supplementation. Increased blood flow to the muscles can decrease within the following 30 min after exercise [32], although the magnitude of the postexercise hyperaemia is proportional to the strength of the contraction and its duration [33]. Depending on the modality, intensity, and duration of the exercise (i.e., muscle contraction), vasodilation may continue for up to 120 min post-exercise. Taken together, post-exercise supplementation may not benefit from exercise-induced muscle blood flow to the same

extent as pre-exercise supplementation due to a shorter overlap between circulating Cr and exercise-induced hyperaemia (Figure 1, Panel B).

Thus, if the primary mechanism by which exercise induces an increased Cr loading of muscle is via an exercise-stimulated increase in blood flow to the working muscles, then preexercise Cr supplementation would be expected to be the most effective supplementation strategy compared to supplementation intra- or post-workout, or at any other moment of the day.

#### *3.2. Na+-K+ Pump Activity and Exercise*

Creatine transport into muscle cells is performed by a specific Cr transporter, CreaT [34]. This transport occurs against a concentration gradient and is dependent on the presence of extracellular Na<sup>+</sup> [35], meaning Cr uptake is achieved via a Na+-Cr cotransport system, which makes use of the sarcolemmal Na+-K+ pumps [36]. Thus, one other mechanism that might optimise Cr supplementation is an upregulation of the kinetics involved in the Cr transport from the bloodstream to the skeletal muscle, via an increase in Na+-K+ pump activity during and following exercise [37]. Indeed, exercise training involving a 2-h exercise cycle per day, for 6 consecutive days at 65% of maximal aerobic power, induced upregulation in sarcolemmal Na+-K+-ATPase concentration in humans, after only one week of training, in the exercised muscle [38].

Studies have shown that the Na+-K+ pump regulates transsarcolemmal [Na+] and [K+] gradients in skeletal muscles and is critical for the maintenance of membrane excitability and contractility [20,39]. Odoom et al. [36] showed that the pharmacological activation and inhibition of Na+-K+ pump activity in mouse myoblast cells were paralleled by up- and downregulation of cellular Cr accumulation, demonstrating the relationship between Na+- K+ pump activity and Cr uptake. This mechanistic evidence suggests that increased Na+-K+ pump activity, as occurs with exercise, might lead to increased Cr uptake (Figure 1A), although it is currently speculative as to whether it occurs in humans.

Since the upregulation of muscle Na+-K+ pump function in the exercised limb facilitates muscle Cr transport, if this mechanism holds true for humans, the timing of Cr supplementation around exercise could alter the uptake into the muscle. Specifically, preexercise supplementation might ensure that high circulating levels of Cr coincide with peak activation of the Na+-K+ pump during exercise-induced muscle contraction, leading to greater intramuscular Cr accumulation (Figure 1B). However, there is a residual effect of exercise that could last from several minutes up to 48 h post-exercise, depending on the action of interest (e.g., insulin sensitivity; [40]), meaning that post-exercise Cr supplementation might also benefit from a contraction-induced potentiation of Na+-K+ pump activity, although this is highly speculative. Furthermore, most individuals taking Cr supplements undertake regular exercise training, which chronically upregulates Na+-K+ pump activity [38]. Thus, it is possible that timing in relation to each exercise session may not be important, but that exercise training in general leads to greater Cr accumulation in muscles due to chronic adaptations in Na+-K+ pump activity. This is somewhat in contrast to evidence suggesting that ingestion of Cr close to an exercise session leads to greater increases in TCr than supplementation that is not close to the exercise session (>5 h) [41].

Skeletal muscles appear to be highly amenable to Cr supplementation, while chronic exercise appears to further increase the response to supplementation. Nonetheless, what is unclear is whether timing around exercise also generates differential responses in the muscle Cr loading response, and subsequent performance gains. The hypothetical mechanisms discussed provide some support to suggest that exact timing, in relation to exercise, may exert differential effects. The following section will detail studies that have investigated the effects of pre-, during-, or post-workout Cr supplementation on several outcomes.

#### **4. Creatine Supplementation Pre-, During- or Post-Workout: The Evidence**

Cribb and Hayes [41] investigated the effects of Cr supplement timing during 10-weeks of resistance exercise training on intramuscular TCr content, muscle-fibre hypertrophy,

strength, and body composition. Recreational male bodybuilders were allocated into two groups: one that consumed their supplements immediately pre- and post-workout on training days, and the other that consumed their supplements in the morning before breakfast and late evening before sleep. The supplements contained 40 g glucose, 40 g protein, <0.5 g of fat, and 7 g of Cr monohydrate per 100 g; participants consumed 1 g·kg−1·day−<sup>1</sup> twice on training days only. The 10-week training program was performed 4 times a week and was designed specifically to increase strength and muscle size, with a progressive overload consisting of three compound exercises with free weights based on repetition maximum (RM) of the participants. The group who ingested the supplements around their workouts had greater increases in intramuscular TCr and greater gains in maximum dynamic strength, lean mass, and cross-sectional area type II fibres compared to the group who consumed Cr at alternate times of the day. These findings suggest that supplement timing can play an important role in strength and muscle gains, although the strength and muscle gains cannot be limited to Cr supplementation only, since the supplement contained various other ingredients, including a substantial amount of protein. It is known that the timing of protein ingestion around exercise may influence hypertrophy and strength gains [42]. Furthermore, carbohydrates were included in the supplement, which was shown to enhance Cr uptake into the muscle [18]; thus, the isolated effect of exercise on muscle TCr loading was not determined. Finally, supplementation was provided, both pre- and post-exercise, as well as "not close" to the exercise session, meaning no inferences can be made regarding whether supplementation pre-, during-, or post-exercise influences these responses.

Timing of Cr supplementation in relation to exercise has been suggested to influence the accrual of muscle Cr [43], which may impact subsequent performance gains. We have discussed the physiological mechanisms through which Cr timing around exercise might modify its loading effects, but it is important to determine the true impact of timing experimentally. A few studies have investigated the influence of supplement timing with Cr in relation to exercise on a number of different outcomes.