Non-enzymatic hydrolysis of creatine ethyl ester

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Abstract

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 °C over the pH range of 1.6–7.0 using 1H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE+), and the diprotonated form (H2CEE2+). The values of pKa1 and pKa2 of H2CEE2+ were found to be 2.30 and 5.25, respectively. The rate law is found to beRate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-]where the rate constants k++, k+, and k0 are (3.9 ± 0.2) × 106 L mol−1 s−1, (3.3 ± 0.5) × 104 L mol−1 s−1, and (4.9 ± 0.3) × 104 L mol−1 s−1, respectively. Calculations performed at the density functional theory level support the hypothesis that the similarity in the values of k+ and k0 results from intramolecular hydrogen bonding that plays a crucial role. This study indicates that the half-life of CEE in blood is on the order of one minute, suggesting that CEE may hydrolyze too quickly to reach muscle cells in its ester form.

Introduction

The efficacy of using creatine as a nutritional supplement for the development of lean muscle mass and strength in athletes has been thoroughly studied and well documented [1], [2], [3], [4], [5], [6], [7]. Based on these results, creatine is used widely in the athletic community. However, the bioavailability of creatine taken as a dietary supplement is limited because of its relatively poor absorption properties [8], [9]. Furthermore, work by Schedel and coworkers shows that creatine levels in the blood stream reach a maximum of about 2.2 mM approximately 2.5 h after the ingestion of a single 20-g oral dose of creatine [10] indicating very slow absorption. As a result, users of the supplement tend to ingest large doses of creatine in order to attain the desired benefit.

Several patents for the synthesis of creatine ethyl ester (CEE) have been issued recently. Within the patents, it has been suggested that the bioavailability of creatine is improved when creatine ethyl ester is ingested instead of creatine [11], [12]. The rationale presented in these patents is that functionalizing the carboxylic acid to the ethyl ester will decrease the “polarity” of the molecule thereby facilitating the transfer of the molecule across cellular membranes. In a recent article, appearing in a popular magazine commonly read by weightlifters and bodybuilders the science editor discussed “new forms of creatine” available to athletes. The author writes, “Because CEE can be absorbed directly into muscle cells, it doesn’t need to rely on insulin and it doesn’t sit outside the muscle cells, which can cause bloating [13]”. Several manufacturers of nutritional supplements now sell CEE even though, as they indicate, their suppositions “have not been evaluated by the Food and Drug Administration”.

An underlying assumption of the claim that CEE can be absorbed into muscle cells more readily than creatine is that the ester can reach muscles without undergoing hydrolysis. However, the rate of hydrolysis of CEE to creatine and ethanol has not been studied. Clearly the rate of hydrolysis of CEE is a fundamental consideration in the evaluation of the claims that it is a better nutritional supplement than creatine.

Half a century ago, the kinetics of the hydrolysis of the esters of some amino acids was studied and the rate constants reported [14]. In that study, the kinetics of the alkaline hydrolysis of nine amino acid esters at 25° and an ionic strength of 0.1 M in water over a pH range from 8.7 to 11.6 was reported. The rate law was found to be of the formRate=-dCAdt=k+/-[HA+][OH-]+k0/-[A][OH-]where [A] represents the concentration of the unprotonated form of the amino acid ester, [HA+] represents the protonated form of the amino acid ester and CA represents the total concentration of the amino acid ester in both forms. The pKa of the amino acid esters without ionizable side chains ranged from 7 to 8. The values of k+/− and k0/− for histidine methyl ester, leucine ethyl ester and glycine methyl ester were found to be 4 × 104 and 4.0 × 101; 5.0 × 104 and 3.5 × 101, and 3.5 × 104 and 7.9 × 101, respectively. In each of these cases, k+/− is about two orders of magnitude greater than that of ko/−. This is not unexpected as k+/− represents a process involving the coming together of a cation and the hydroxide anion, while k0/− represents the coming together of a neutral species with hydroxide ion. (Over the pH range studied, the histidine side chain remains essentially unprotonated.) In addition to the simple base (OH)-catalyzed hydrolysis of these esters, ester hydrolysis may also be catalyzed by enzymes [15], [16].

Based on the non-enzymatic hydrolysis rate constants for the amino acid esters reported by Hay and coworkers [14], the half-lives of the amino acid esters at a physiological pH of 7.4 would be on the order of 10 h. Should CEE show the same or greater level of stability, then further experiments determining its efficacy would be of value. On the other hand, if the rate of hydrolysis is fast, then the issue of bioavailability becomes moot. It is the non-enzymatic hydrolysis of CEE that is the subject of this study. The objective is to determine the likelihood that CEE taken orally could make its way to skeletal muscle cells before being hydrolyzed to creatine and ethanol.

Section snippets

Materials and methods

Reagents. Commercially available creatine ethyl ester hydrochloride was obtained from Higher Power Nutrition. NMR experiments showed no impurities in the creatine ethyl ester hydrochloride, and it was deemed to be sufficiently pure to be used without further purification. Solutions of sodium hydroxide were prepared by appropriate dilution from 50% NaOH obtained from VWR. The dilute solutions were standardized by titration of dry, certified potassium hydrogen phthalate obtained from Fisher

Acid–base properties

That CEE can undergo two protonation steps in aqueous solution can be seen clearly in Fig. 1. Based on the concentrations and volumes used in the titration the values of pKa1 and pKa2 were found to be 2.30 and 5.25, respectively. To identify the most likely sites of protonation, CEE was modeled and the potential surface at a value of −160 kJ mol−1 was mapped. The results, shown in Fig. 2, are for two conformations: the lowest energy conformer (in which there is significant intramolecular hydrogen

Discussion

As with free creatine, the first site of protonation (the most basic site) of CEE is the guanidinium functionality. However, the pKa for the deprotonation of this site (5.25 at 25 °C) is seven pKa units smaller than that reported by Wang et al. for free creatine (12.90 at 30 °C) [19]. This difference suggests that the free energy change for the transfer of a proton from the protonated guanidinium functionality of creatine is about 40 kJ mol−1 more positive than that for the transfer of a proton

Acknowledgments

This research was supported by the Chemistry Summer Undergraduate Research Program, the Office of Research, and the Department of Chemistry and Biochemistry at The University of Tulsa.

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    This work was presented in part at the 237th ACS National Meeting in Salt Lake City, UT, March 25, 2009.

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