Kinetic Analysis of the Lactate-dehydrogenase-coupled Reaction Process and Measurement of Alanine Transaminase by an Integration Strategy

Abstract

Kinetic analyses of lactate-dehydrogenase (LD)-coupled alanine transaminase (ALT) reaction processes were investigated for measuring ALT by an integration strategy. For measuring ALT by a kinetic analysis of an LD-coupled ALT reaction curve, candidate reaction curves were calculated via iterative numerical integration of the differential velocity equations to execute a weighted nonlinear-least-square-fitting. To realize the integration strategy, the conventional initial-velocity method was used if the ALT activities were below 25 U/L; otherwise, kinetic analyses of the reaction curves were employed. Of the reaction curves recorded at 10-s intervals, kinetic analyses gave ALT activities resistant to deviations in the LD kinetic parameters. The integration strategy yielded a higher value of the lower limit, but an upper limit of over 100 U/L by simulations and over 75 U/L with purified ALT. Also, its intra-run relative standard deviations were below 9% for 0.50 U/L ALT and below 5% for final 1 to 65 U/L ALT. The integration strategy gave consistent ALT activities in clinical sera. Hence, this new approach for kinetic analyses of ALT reaction processes and the integration strategy were effective to measure ALT.