Studies of electrophoretic detection methods for serum creatine kinase isoenzymes

Abstract

This study is concerned with the activity of creatine kinase isoenzymes through electrophoretic detection techniques. It is well documented that creatine kinase may be elevated in a variety of disease states. However, the only concern of the study was with CK elevations due to cardiac involvement, namely acute myocardial infarction.

The initial step was devoted to two methods for the determination of total creatine kinase activity. These two methods included; (1) the kinetic method from the Boehringer Mannheim Corporation (BMC), and the luciferin-luciferase method from Antonik Laboratories. Once the total activity was determined, a standard curve was then developed using control sera from Helena Laboratories.

Completing this phase of the study, it was then necessary to turn all the attention to electrophoresis. Commercially prepared control sera from various manufacturers were obtained and examined for their potential use as creatine kinase isoenzyme markers. The electrophoretic setup used for the analysis was from Helena Laboratories, and the CK substrate was from BMC. It was discovered that no commercially prepared chemistry control sera could be utilized as a marker for CK isoenzymes, unless a particular control was sold specifically for this purpose.

Another phase of this study involved the reexamination of cord blood sera as potential markers for CK isoenzyme activity. Various cord blood samples were obtained from the maternity wards of area hospitals and examine for their creatine kinase isoenzyme content. It was hoped that the cord blood specimens would contain all three isoenzyme fractions so that the expense of purchasing commercially prepared CK isoenzyme control serum could be alleviated. Ten cord specimens were examined by the fluorometric method of Helena, and only MM and BB fractions were demonstrated.

Migration studies of the CK isoenzymes were then examined in their relationship to serum protein electrophoresis. It was shown that the MM fraction migrated with the gamma globulin fraction, BB migrated with albumin fraction and MB migrated with the alpha-2 globulin fraction of total protein. Again the electrophoretic techniques, as outlined by Helena Laboratories were employed.

The final phase of this study involved a comparative examination of various sera form patients diagnosed as having had an acute myocardial infarction by two techniques. These techniques involved the method by Helena, and a bioluminescent method utilizing ATP, luciferin, and luciferase. The established Helena procedure demonstrated excellent results, with MB fractions of low activity being readily detected by this method. However when luciferin-luciferase system was used, much difficulty was encountered. Most of the problems stemmed from diffusion of the protein molecules during development. In an attempt to correct this diffusion problem, various buffers and solutions were adjusted, optimized conditions were sought, and alterations in technique were employed. However all endeavors proved futile.

Through this study however, much knowledge of the creatine kinase isoenzymes were obtained, as to their activity, mobility, clinical usefulness and diagnostic importance relating to myocardial infarction.