Flow-injection fluorometric quantification of pyruvate using co-immobilized pyruvate decarboxylase and aldehyde dehydrogenase reactor: Application to measurement of acetate, citrate and L-lactate

摘要

A flow-injection system for the quantification of pyruvate based on the coupled reactions of pyruvate decarboxylase (PDC) and aldehyde dehydrogenase (AlDH) was conceived and optimized. A co-immobilized PDC and AIDH reactor was introduced into the flow line. Sample and reagent (NAD+) were injected into the flow line by an open sandwich method and the increase of NADH produced by the immobilized-enzyme reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of pyruvate were observed in the ranges of 2.0 x 10(-5) to 1.5 x 10(-3) M at the flow rate of 1.0 ml min(-1) and 5.0 x 10(-6) to 1.0 x 10(-3) M at the flow rate of 0.5 ml min(-1). The relative standard deviation for 10 successive injections was 0.95% at the 1.0 mM level. This FIA system for pyruvate was applied to the measurement of acetate, citrate and L-lactate. (c) 2005 Elsevier B.V. All rights reserved.