ENZYMATIC METHODS FOR THE DETERMINATION OF ALPHA-GLYCEROPHOSPHATE ANDALPHA-GLYCEROPHOSPHATE OXIDASE WITH AN AUTOMATED FIA SYSTEM

A procedure for the enzymatic determination of alpha-glycerophosphate (alpha-GP) has been developed, using an automated in-house FIA system, with immobilized glycerol-3-phosphate oxidase (GPO) on non-porous gla ss beads, following optimization of the immobilization and analytical parameters. Fabricated single bead string reactors (SBSR) were used in connection with the FIA system, following optimization of its paramet ers. The half-life of GPO-SBSR regarding reduction of the enzyme activ ity was found to be 110 days for its use in 20 triplicate measurements daily and storage at 4 degrees C in the appropriate buffer. The regre ssion equation of the calibration graph for the determination of alpha -GP was: A(max) = (10 +/- 2) x 10(-4) + (22134 +/- 12) x 10(-4) (mmol l(-1) alpha-GP). The lower limit of quantitation was 0.74 mu mol l(-1) alpha-GP and the RSD of the method 0.05% (r = 0.9999). The same FIA s ystem and procedure can be also used for the determination of the GPO activity, with the alpha-GP as substrate. The regression equation for this calibration graph was: A(max) = (23 +/- 18) x 10(-4) + (190 +/- 1 ) x 10(-4) (mu g ml(-1) GPO), the lower limit of quantitation was 0.78 2 x 10(-3) mg ml(-1) (0.782 ppm) GPO and the RSD of the method 0.53% ( r = 0.9999). Serum samples obtained from hospitalized patients were de proteinized by gel filtration and analyzed under pseudo-first order co nditions, at various concentrations of alpha-GP. A kinetic study of th e reduction of alpha-GP in serum versus time is given and an observed reaction rate constant k(ob) = 106.5 x 10(-4) min(-1) was determined. (C) 1998 Elsevier Science B.V.