AutomatedFluorometricProcedurefor Measurement of CreatinePhosphokinaseActivity

An automated procedure is described for measur-ing creatine phosphokinase activity (in Serum, although the technique is adaptable) with the Technicon AutoAnalyzer. Creatine, formed by the action of the enzyme on phosphocreatine and adenosine diphosphate, is measured by the fluorescence of its reaction product with alkaline ninhydrin solution. Thirty determinations can be made per hour, and only 0.2 ml of sample is re-quired. Additional Keyphrases AutoAnalyzer #{149} alkaline ninhydrin and guanidyl compounds, reaction #{149} ultra-violet manual method, results compared #{149} normal values for serum Creatine phosphokinase (cPK; ATP: creatine phosphotransferase, EC 2.7.3.2) activity may be measured by various manual procedures (1-7), the most sensitive of which (4, 6, 7) measure formation of ATP or creatine from ADP and phosphocreatine in the reaction: cPK ADP + phosphocreatine ATP + creatine In 1959, Conn and Davis (8) showed that crea-tine and other guanidyl compounds react with ninhydrin in alkaline solution to give fluorophors, whereas phosphocreatine reacts to a much lesser extent. This reaction was subsequently used to determine creatine (9) and in the manual deter-mination of CPK (5, 6). We describe here an adapta-tion of the reaction to the fully automated deter-mination of CPK. Materials and Methods Principle Creatine formed by the action of creatine phos-phokinase on ADP and phosphocreatine reacts with alkaline ninhydrin solution to give a fluores-cent product. The fluorescence is measured by use of a fluorometer connected to the AutoAnalyze