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

提出了一种快速测定动脉血酮体浓度和动脉血比（AKBR）的新方法。该方法涉及在β -羟基丁酸脱氢酶存在的情况下，由NAD^+生成NADH，与β -羟基丁酸的浓度成比例。在NADH氧化酶存在的情况下，NADH与氧结合产生过氧化氢。在过氧化物酶存在下，3-甲基-2-苯并噻唑啉-腙-6-硫酸盐（MBTH-S）和n -乙基- n -磺丙基-3,5维苯胺（MAPS）氧化偶联反应生成红紫色产物。-羟基丁酸盐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，将乙酰乙酸酯完全转化为β -羟基丁酸酯，然后测定β -羟基丁酸酯的总浓度，从而测定乙酰乙酸酯的浓度。乙乙酸浓度由酮体总浓度减去-羟基丁酸浓度得到。为了获得更高的灵敏度，我们一直在研究作为耦合器的MBTH- s（或MBTH类似物）与Trinder试剂之间的反应条件。MBTH模拟；2-[（甲基磺酰基）肼]-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（pH 8.0）中，MBTH-S和MAPS的灵敏度是当前条件下的2倍，但试剂空白增加较大，影响了重现性。我们正在研究改进它的方法。A和B的灵敏度和稳定性都不如MBTH-S。

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.