Study of the heterogeneity of creatine kinase-MM isoforms using Western blotting

Abstract

To study creatine kinase isoform variants in muscle (skeletal and myocardial) damage using a Western blotting procedure. The study comprised of 16 patients admitted with chest pain, 14 patients with skeletal muscle damage, and 4 healthy individuals. The creatine kinase-MM (CK-MM) isoforms were separated by isoelectric focusing and detected by electrophoretic transfer to a nitrocellulose membrane and immunoblotting. Binding of the first monoclonal mouse anti-human CK-MM antibody was detected by either a horseradish peroxidase or alkaline phosphatase anti-mouse immunoglobulin conjugate. Three major variants of CK-MM (MM3 pl = 6.8 MM2 pl = 6.4; MM1 pl = 6.2) were detected. Minor sub-bands were detected in 4/16 and 3/14 patients with acute myocardial infarction (AMI) and skeletal muscle damage, respectively. The tissue form (MM0 pl = 7.14) was visible in 6/16 patients with AMI and 4/14 patients with skeletal muscle damage. The appearance of minor variants was therefore of limited diagnostic value in discriminating between patients with and without AMI. Immuno-blotting confirms the parallel increase in mass and activity of CK-MM isoforms, thus confirming measurement of isoform activity as an early marker of AMI or reperfusion following thrombolytic therapy. Study of isoform patterns gives more information concerning AMI and skeletal muscle trauma than is currently available from CK isoenzyme analysis. However, because CK-MM is also released in skeletal muscle damage, in such instances more discriminant markers of AMI are needed.