QualitativeIn vitroNMR Analysis of Creatine Ethyl Ester Pronutrient in Human Plasma

Abstract

There are a number of forms of creatine available that attempt to improve the solubility and permeability, with the anticipation this will result in an improved pharmacokinetic profile and ultimately an enhanced ergogenic response. Previous research has shown that the different salt forms can improve solubility resulting in slightly altered pharmacokinetic profiles, however specific data exploring the conversion of esterified derivatives to creatine is lacking. The purpose of this study was to examine the assertion that creatine ethyl ester undergoes enzymatic conversion to creatine in human tissues. The IN VITRO response of creatine ethyl ester to incubation in human plasma was examined by H-NMR analysis. Lyophilized human plasma was reconstituted in D2O and phosphate-buffered saline and 1.5 mg of the analyte was added. Following incubation at 37 degrees C for 4 h and subsequent protein precipitation, the supernatant was analyzed by NMR, utilizing the diagnostic chemical shift of the methylene signal to determine the species present in solution, I.E. creatine ethyl ester, creatine, or creatinine. Both creatine and creatinine were run in parallel as control experiments and each assay was run in triplicate. As expected both creatine and creatinine remained unchanged. However, conversion of creatine ethyl ester to creatine by the esterases in human plasma was not observed to any detectable extent and the only species detected after the incubation period was creatinine. While not a definitive characterization of the IN VIVO behavior, these results strongly warrant a complete IN VIVO pharmacokinetic analysis of creatine ethyl ester since it appears these "pronutrients" may actually provide large exogenous sources of pharmacologically inactive creatinine rather than ergogenic creatine.

pH-Dependent Stability of Creatine Ethyl Ester: Relevance to Oral Absorption

“…Despite a previous qualitative NMR study describing an esterase-mediated degradation of CEE to CRN in human plasma (Giese and Lecher, 2009b), we suggest that the degradation of CEE does not depend on esterases. First, the product of CEE degradation in plasma is CRN, not CRT, and second, the degradation rate in plasma is similar to that in aqueous solution of equal pH.…”

“…The latter intramolecular reaction likely accounts for the undesired formation of CRN hydrochloride observed in the equilibrium esterification process used for the synthesis of CEE from CRT monohydrate (Mold, Gore, Lynch, & Schantz, 1955; Vennerstrom, 2005). Recent experiments (Giese & Lecher, 2009a, 2009b; Katseres, Reading, Shayya, DiCesare, & Purser, 2009) using proton NMR in deuterated water suggest that CEE is converted to CRN with a half-life of less than one min at pH 7.4 in the presence and absence of human plasma, although CEE solution stability increased considerably as pH decreased. This contradicts a report (Child & Tallon, 2007) that CEE undergoes significant conversion to CRN in low pH environments, suggesting that the degradation of CEE in the stomach would limit oral bioavailability.…”

Analysis of the efficacy, safety, and regulatory status of novel forms of creatine

“…These findings are also in accordance with the recent investigations on the stability of CEE at 37ºC in both water and phosphate-buffered saline and the in vitro response of CEE to incubation in human plasma by H-NMR analysis (Giese and Lecher 2009b). The conversion of CEE to creatine by the esterases in human plasma was not detected, and the only species detected after the incubation period was creatinine.…”

Authorised EU health claims for creatine

“…A separate study compared the stability of CM and CEE in blood plasma using nuclear magnetic resonance (Giese and Lecher, 2009b). This also demonstrated greater conversion of CEE to creatinine, leading the authors to conclude 'it appears these "pronutrients" may actually provide large exogenous sources of pharmacologically inactive creatinine rather than ergogenic creatine'.…”

“…These differences in metabolism would be expected to have a significant impact on the ability of commercial creatine products to deliver physical performance benefits (Giese and Lecher, 2009b). The metabolic fates of orally ingested CM and CEE are outlined in Fig.…”