New manual and automated method for determining activity of creatine kinase isoenzyme MB, by use of dithiothreitol: clinical applications.
The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.
D000818:Animals; D001322:Autoanalysis; D003402:Creatine Kinase / Q000097:blood* / Q000302:isolation & purification; D004229:Dithiothreitol / Q000494:pharmacology*; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D005069:Evaluation Studies as Topic; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D009206:Myocardium / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D007527:Isoenzymes; D003402:Creatine Kinase; D005978:Glutathione; D004229:Dithiothreitol