Clinical Chemistry, Vol 34, 235-239, Copyright © 1988 by American Association for Clinical Chemistry

Quantitative analysis for isoforms of creatine kinase MM in plasma by chromatofocusing, with on-line monitoring of enzyme activity

R Nohara, BE Sobel, AS Jaffe and DR Abendschein
Cardiovascular Division, Washington University School of Medicine, St. Louis, MO 63110.

Changes in the proportions of individual isoforms of the MM isoenzyme of creatine kinase (CK; EC 2.7.3.2) in plasma promptly reflect both myocardial infarction and coronary recanalization. However, quantitative methods developed thus far are too slow or cumbersome for routine use in making clinical decisions. We report a convenient, quantitative chromatofocusing assay with on-line fluorometric detection of isoform activity in the column eluent that provides results within 40 min from the time of sample application. Sample eluted from a microbore chromatofocusing column (1.8-mL bed volume) is split between a reaction stream, into which CK reagents are added, and a reference stream. After incubation at 37 degrees C, NADPH formed by reaction of isoforms with CK reagent is detected at 340 nm. The system can detect activity of individual isoforms in plasma samples having total CK activity greater than or equal to 21 U/L (30 degrees C). Results correlated closely with those obtained by previously validated, but slow, chromatofocusing (r = 0.98, n = 30) and protein immunoblotting (r = 0.90, n = 20) procedures.