Creatine synthesis in isolated hepatocytes

Abstract

Creatine (Cr) is a “high-energy” phosphate buffer that is synthesized from glycine, arginine and methionine. The generally accepted pathway for Cr synthesis involves both the kidney and liver. Guanidinoacetate (GAA) is formed from glycine and arginine by the enzyme arginine:glycine amidinotransferase (AGAT) in the kidney. GAA is then transported to the liver where it is methylated by the enzyme guanidinoacetate methyltransferase (GAMT) to form Cr. Cr is also spontaneously, non-enzymatically cyclized to creatinine(Crn), which is subsequently excreted in the urine. Daily loss of creatine is on the order of 1-2% of the total body pool and it has been calculated that the replacement of lost Cr would require about 50% of daily arginine intake and 70% of daily methionine intake. While methionine can be recycled after methylation, the ornithine produced from arginine is largely oxidized in the kidney. However, if AGAT activity was present in the liver the ornithine produced could re-enter the urea cycle and thus recycle arginine. It is the goal of this study to determine whether this occurs in rat hepatocytes. In addition, little is known about the transport of GAA and Cr in the liver and part of the goal of this study will be to investigate the transport of these compounds. We measured Cr synthesis in isolated hepatocytes using various substrates. When GAA and methionine were present Cr synthesis was determined to be 0.02 nmol/min/mg dry weight. Cr synthesis was inhibited by 46% in the presence of 3-guanidinopropionic acid; a known inhibitor of Cr transport. Cr did not appear to be synthesized in the presence of NH4, arginine, glycine and methionine and therefore the entire synthetic pathway probably does not occur in rat hepatocytes. Supported by CIHR.