High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues beta-guanidopropionic acid and 1-carboxy-methyl-2-iminoimidazolidine (cyclocreatine).

Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.