A study on a sensitive assay of AKBR for rapid evaluation of pathophysiological state of liver

去年发展了一种新的快速测量动脉血酮体浓度和测定动脉血比(AKBR)的方法。该方法涉及在β-羟基丁酸脱氢酶存在下，由NAD^生成与β-羟基丁酸浓度成比例的NADH。在NADH氧化酶的存在下，NADH与氧气反应生成过氧化氢。在过氧化物酶存在下，3-methyl-2-benzothiazolinone-hydrazone-6-sulfate与N-乙基-N-磺丙基-3，5-二甲基苯胺(MAP)发生氧化偶联反应，生成紫红色产物。β-羟基丁酸酯的浓度是通过测量颜色强度的增加来确定的。在β-羟基丁酸脱氢酶存在下，乙酰乙酸酯完全转化为β-羟基丁酸酯，然后测定总的β-羟基丁酸酯浓度，从而确定乙酰乙酸酯的浓度。乙酰乙酸酯浓度是用酮体总浓度减去β-羟基丁酸酯浓度得到的。为了获得更高的灵敏度，我们一直在研究偶联剂MBTH-S(或MBTH类似物)与特林德试剂的反应条件。2-[(甲磺酰基)肼]-2，3-dihydro-3-methyl-6-benzothiazole-sulfonic酸(A)2-hydrazone-2，3-dihydro-3-methyl-6-benzo-thiazolesulfonyl氨基乙酸(B)结果在此缓冲液(pH 8.0)中使用MBTH-S和MAP的灵敏度比目前条件下提高了一倍，但试剂空白的增加较大，这对重现性有很大的影响。我们正在研究改进的方法。A和B都不比MBTH-S有更高的灵敏度或稳定性。

A new rapid method for the measurement of arterial blood ketone body concentration and determination of arterial blood ratio (AKBR) was developed last year. This method involves the generation of NADH from NAD^+ in proportion to the concentration of beta-hydroxybutyrate in the presence of beta-hydroxybutyrate dehydrogenase. NADH is reached with oxygen in the presence of NADH oxidase to produce hydrogen peroxide. The oxidative coupling reaction of 3-methyl-2-benzothiazolinone-hydrazone-6-sulfate (MBTH-S) and N-ethyl-N-sulfopropyl-3,5-dimenthylaniline (MAPS) produces a red-purple product in the presence of peroxidase. The concentration of beta-hydroxybutyrate is determined by measuring the increase in intensity of the color. The acetoacetate concentration is determined by completely converting it to beta-hydroxybutyrate, in the presence of beta-hydroxybutyrate dehydrogenase, and then assaying the total beta-hydroxybutyrate concentration. The acetoacetate concentration is obtained by subtraction of beta-hydroxybutyrate concentration from the total ketone body concentration. We have been studying reaction conditions between MBTH-S (or MBTH analog) as a coupler and Trinder's reagent to get a higher sensitivity. MBTH analog ; 2- [(methylsulfonyl) hydrazono] -2,3-dihydro-3-methyl-6-benzothiazole-sulfonic acid (A) 2-hydrazone-2,3-dihydro-3-methyl-6-benzo-thiazolesulfonyl aminoacetic acid (B)Results Sensitivity using MBTH-S and MAPS in this buffer (pH 8.0) became twofold as high as the one in the current condition, though the increase of the reagent blank was larger, which had a bad influence on the reproducibility. We are studying the method to improve it. Neither A nor B brought higher sensitivity or stability than MBTH-S.