Chromatographic separation and continuously referenced, on-line monitoring of creatine kinase isoenzymes by use of an immobilized-enzyme microreactor.

We describe a new concept in continuously referenced monitoring of the isoenzyme activities of creatine kinase (EC 2.7.3.2) after liquid-chromatographic separation. After separation on a diethylaminoethyl-Sephacel column, the three isoenzymes of creatine kinase undergo a series of coupled enzyme reactions, ultimately resulting in the formation of ultraviolet-detectable NADPH. A major advantage of this detection system is the immobilized-enzyme microreactor (2 X 17 mm), which may be removed and stored refrigerated when not in use. A split-stream configuation allows self-blanking of endogenous ultraviolet-absorbing constituents in authentic sera samples, which would otherwise make definitive diagnosis and quantitation difficult or impossible. This system is applicable to the automated analysis of creatine kinase isoenzymes in the clinical laboratory.