Continuous assay of serum 5-nucleotidase activity with inosine 5'-monophosphate as substrate and total automation...

The continuous spectrophotometric assay of 5'-nucleotidase originally described by Heinz et al. ((1980) J. Clin. Chem. Clin. Biochem. 18, 781-788) was modified and fully automated on a Kem-O-Mat transfer analyzer, using inosine 5'-monophosphate as substrate. The reaction product was hydrogen peroxide and the reduction of NADP was observed for 10 minutes at 340 nm and at a reaction temperature of 30 degrees C. The different factors involved in the enzyme reaction were checked, including the substrate concentration, reaction rate, linearity and substrate preservation. Normal values ranged from 1 to 13 U/l. Between-day reproducibility was estimated with two different commercial control sera, and the coefficient of variation was 5% for the upper limit of normal activity (23 U/l). There was good agreement between the present method and a semi-automatic colorimetric technique (for 100 sera tested by both methods, the correlation coefficient was 0.974 and the regression line equation, y = 0.85 x- 1.5). Despite the lengthy reagent mixture preparation procedure, the method permitted assay of 50 samples per hour. The occurrence of high serum blanks in certain pathological states is discussed.