Determination of Isocitrate Using Immobilized Isocitrate Dehydrogenase in a Flow System and its Application to Analyze the Total Isocitrate Content of Beverages

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The quantity of isocitrate was determined using an apparatus containing a reactor with immobilized isocitrate dehydrogenase in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD+ in the carrier was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of Tris buffer ranged from 6.0 to 8.6. Various buffer types were also examined as carrier mediums at pH 8.0. In contrast to 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and triethanolamine buffers which afforded comparable peak areas with that of Tris buffer, phosphate buffer showed a reduced peak area. This peak area decreased with an increase in the pH of the phosphate buffer from 6.5 to 8.5, suggesting the inhibitory effect of phosphate dianions upon the binding of adenosine-5′-monophosphate (AMP) to the binding site of the enzyme. When the carrier composed of Tris buffer (0.1 M, pH 8.0) was used, the calibration curve for isocitrate was linear in the range of 0.1-50 μM (r = 1.000). The detection limit (S/N = 3) was 0.07 μM. Relative standard deviations of the peak area at 1 and 10 μM were 4.0% (n = 7) and 2.8% (n = 7), respectively. Thirty samples of isocitrate (2 μM) were analyzed for 1 hr. This method was applied to the analysis of total isocitrate in several beverages. The recovery tests for the isocitrate added to samples indicatied the reliability of the present method.