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定动脉血酮体浓度和测定动脉血比率（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。3-甲基-2-苯并噻唑啉酮-腙-6-硫酸酯（MBTH-S）与N-乙基-N-磺丙基-3，5-二甲基苯胺（MAPS）在过氧化物酶存在下发生氧化偶联反应，生成紫红色产物。通过测量颜色强度的增加来确定β-羟基丁酸酯的浓度。通过在β-羟基丁酸脱氢酶存在下将其完全转化为β-羟基丁酸，然后测定总β-羟基丁酸浓度，测定乙酰乙酸浓度。通过从总酮体浓度中减去β-羟基丁酸酯浓度获得乙酰乙酸酯浓度。为了获得更高的灵敏度，我们研究了MBTH-S（或MBTH类似物）作为偶联剂与Trinder试剂的反应条件。MBTH类似物; 2- [（甲磺酰基）亚肼基]-2，3-二氢-3-甲基-6-苯并噻唑磺酸（A）2-腙-2，3-二氢-3-甲基-6-苯并噻唑磺酰基氨基乙酸（B）结果在该缓冲液中使用MBTH-S和MAPS的灵敏度（pH 8.0）变为当前条件下的两倍，尽管试剂空白的增加更大，这对再现性有不良影响。A和B的灵敏度和稳定性都没有MBTH-S高。

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.