Development and Application of High-performance Liquid Chromatography/Chemiluminescence Determination of Biological Thiols

本研究取得的主要结果如下：1.建立了以N-[4-（6-二甲基氨基-2-苯并呋喃基）苯基]马来酰亚胺（DBPM）为荧光标记物的高效液相色谱（HPLC）-化学发光（CL）测定生物硫醇的方法，并应用于大鼠肝脏中还原型谷胱甘肽和半胱氨酸的测定。该方法灵敏度高，可检测到fmol水平的硫醇。接下来，开发了使用4-氟-7-硝基苯并-2，1，3-恶二唑（NBD-F）作为标记的另一种HPLC-CL方法。但该方法的灵敏度较低，为pmol级.在本研究过程中，发现对于某些硫醇，CL检测并不总是比荧光检测灵敏。这可能是由于所用发光试剂的适用性所致.为了评价过氧草酸盐化学发光试剂的性能，建立了一种简单、快速的流动体系分析方法。利用该方法对新合成的芳香羧酸酯和荧光化合物进行了评价，发现了一些有用的化学发光试剂。该方法适用于HPLC-过氧草酸盐化学发光试剂的选择.用2法测定了丙二醛（MDA）与硫代巴比妥酸缩合物的化学发光效率。结果表明，MDA-1，3-二乙基-2-硫代巴比妥酸（DETBA）缩合物显示出最大的CL效率，可检测到低至10 fmol。这表明DETBA是一种优良的MDA衍生试剂。DETBA是一种高灵敏度的脂质过氧化物（MDA）检测方法。

The results obtained in this study are follows:1. A high-performance liquid chromatography (HPLC)-chemiluminescence (CL) determination method of biological thiols using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) as a fluorogenic label has been developed and applied to the assay of reduced glutathione and cysteine in rat liver. The method was very sensitive and could detect fmol levels of thiols. Next, another HPLC-CL method using 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole (NBD-F) as a label was developed. However, the sensitivity of the method was lower and pmol level. In the course of this study, it was found that CL detection was not always sensitive than fluorescence detection for some thiols. It might be caused by the suitabilities of the chemiluminogenic reagents used.2. To evaluate oxalates and fluorescers as peroxyoxalate CL reagents, a simple and rapid method by flow system has been developed. By the method, newly synthesized aryl oxalates and fluorescent compounds were evaluated and found some useful compounds as CL reagents. The method should be suitable for the selection of HPLC-peroxyoxalate CL reagents.3. By the method of 2, CL efficiencies of condensates of malondialdehyde (MDA) with thiobarbituric acids were determined. As the result, MDA-1,3-diethyl-2-thiobarbituric acid (DETBA) condensate showed the largest CL efficiency and could be detect as low as 10 fmol. This suggests that DETBA would be an excellent derivatization reagent for MDA. By using DETBA, a high sensitive assay method for lipoperoxide (MDA) should be developed.