Fluorophotometric Determination of Lactate Dehydrogenase and Creatine Phosphokinase Activities with the Pyridoxamine-Zn (II) ion

Abstract

Fluorophotometric assays for serum lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were developed. α-Oxo-acids react with pyridoxamine and zinc (II) ion to produce a characteristic fluorescence (Ex. 395 nm, Em. 475 nm). This fluorescence reaction was applied to the assay of LDH and CPK activity. The method for the assay of LDH activity is based on the determination of pyruvate produced in the incubation mixture containing lactate and nicotinamide adenine dinucleotide. The CPK activity is also determined by fluorimetric assay of pyruvate in the incubation mixture containing creatine, adenosine triphosphate, phosphoenolpyruvate and pyruvate kinase. These methods require only 5-10 μl of serum sample. The values obtained by these methods satisfactorily correlate with those obtained by the ordinary ultraviolet methods and showed good reproducibility. The correlation coefficients are 0.960 for LDH and 0.965 for CPK, respectively. The method for CPK could be applied to the assay of CPK isoenzymes.