Amperometric flow-injection analysis of creatinine based on immobilized creatinine deiminase, leucine dehydrogenase and L-amino acid oxidase

Abstract

Leucine dehydrogenase/ l-amino acid oxidase was proposed as an enzymatic conversion system for ammonia and its application to amperometric assay of creatinine was investigated. Ammonia formed by creatinine deiminase catalyzed hydrolysis of creatinine was converted to l-leucine by leucine dehydrogenase, and the oxidation of l-leucine by l-amino acid oxidase was detected with an oxygen electrode. Two approaches were proposed to overcome the problem of endogenous ammonia and l-amino acids. The first was using glutamate dehydrogenase prereactor to remove endogenous ammonia; endogenous l-amino acids were corrected by a separate run. In the second approach, endogenous ammonia and l-amino acids were simultaneously compensated with a two-channel system. It resulted in double peak recording that the flow was split and rejoined between the two ends of creatinine deiminase reactor and a delay coil and a reference column were properly set at one of the two-channels. One gave the sum response of all responsible compounds, the other that of endogenous interferences except creatinine. Both approaches were applied to creatinine assay in urine and the results showed a good agreement with those obtained from the Jaffe method.