Assay Of Alanine α-ketoglutarate Transaminase Activity By Kinetic Analysis Of Its Reaction Coupled To Reaction Of Lactic Acid Dehydrogenase

Different methods were compared to measure activity of alanineα-ketoglutarate aminotransferase (ALT) via coupled reaction to action of lactic acid dehydrogenase (LDH), including the classical initial rate method, an integration method of kinetic analysis of LDH-coupled reaction with the classical initial rate method, and a maximal instantaneous reaction rate method. The following results were obtained:1) A method to process LDH-coupled ALT reaction process was developed. Based on differential rate equations for non-steady reaction of LDH, a combination of parameters were preset to calculate a simulated ALT-LDH reaction process that was used for least-square fitting to experimental reaction curve; the combination of parameters giving the least square of residuals gave the indexes of a ALT-LDH reaction system.2) Analyses of simulation data supported that the integration method and the maximal instantaneous reaction rate method were superior to the classical initial rate method based on their upper limit for linear response;the maximal instantaneous reaction rate was the best due to its much small computation work when cost of LDH was not a burden, but the integration method was the best when the cost of LDH was a burden except its heavy computation time.3) By analyzing reaction data of purified ALT, it was found that both the integration method and the maximal instantaneous reaction rate method were better than the classical initial rate method based on its upper limit for linear response. By analyzing reaction data with serum samples, it was found that ALT activities in sera by the integration method were positively and closely correlated with those by the classical initial rate method, and were consistent with those by the classical initial rate method via Bland-Altman analysis.Therefore, both the integration method and maximal instantaneous reaction rate method were promising to measure ALT activity in clinic sera via coupled reaction of LDH.