Wiseman R W, Moerland T S, Chase P B, Stuppard R, Kushmerick M J
Department of Radiology, University of Washington Medical Center, Seattle 98195.
Anal Biochem. 1992 Aug 1;204(2):383-9. doi: 10.1016/0003-2697(92)90255-6.
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.
肌酸和磷酸肌酸是肌酸激酶的底物，肌酸激酶是细胞内能量转移过程中的关键酶。已将肌酸类似物喂给动物，以确定该酶在能量代谢中所起的作用，但由于缺乏定量技术来测定这些化合物的组织含量，解释方面的进展受到了阻碍。我们描述了通过高效液相色谱法对取代胍基化合物及其磷酸化形式进行分离和定量的方法。首先，使用阳离子交换柱测定游离肌酸及其未磷酸化的类似物，然后在阴离子交换柱上测定磷酸肌酸及其磷酸化类似物以及腺苷酸含量（AMP、ADP、ATP）。这些方法已被证明在测量哺乳动物骨骼肌中和高氯酸提取物中这些化合物的化学含量方面是成功的。该方法的灵敏度范围为50至200皮摩尔，足以从5至10毫克样品的组织提取物中获取信息。
