基于手性整体柱全自动二维高效液相色谱技术的体内D-型酸性氨基酸的分布及生理机能研究

Since the presence of naturally occurring D-acidic amino acids in mammals has been observed, their physiological importance and roles of disease bio-marks and has gradually become the focus of the discovery of new drugs which act on neuron and endocrine system, as well as the discovery of new diagnostic methods of the neuron and endocrine disease. However, the sensitivity quantitative analysis of D-acidic amino acids and its L- isomers in mammalian tissues and physiological fluids is quite difficult because of interference of large amount L-amino acids and unknown intrinsic substances in the complex biological matrices. How to separate the trace D-acidic amino acids from their L-forms and determine the both of them simultaneously with high speed and selectivity is becoming a problem. In this study, based on the 2D-HPLC methods for the analysis of amino acids established in previous work, we plan to discover some new type of high efficiency monolithic enantiomer separation column and ODS column to establish a new fully-automatic highly selectivity monolithic 2D-HPLC system using multi-loop technique so that the simultaneous determination of trace amounts of D-acidic amino acids and their L- isomers in the biological samples can be carried out with high selectivity. Moreover, by applying this monolithic 2D-HPLC system,we plan to investigate the intrinsic amounts of D- and L-forms of acidic amino acids in the tissues and physiological fluids of D-aspartate oxidase (DDO) knock-out mice with various DDO activities and various kinds of disease models mice and rats. According to the results in this study,the physiological and pharmacological functions and the roles of disease bio-marks of D-acidic amino acis are excepted to be clarified and the results in this study should be useful for designing novel drugs controlling DDO activity to regulate the intrinsic D-acidic amino acids amount for the treatment of neuronal and endocrine diseases or useful for the development of D-acidic amino acids contained dietary supplement and health food.

自D-型酸性氨基酸在哺乳动物体内的微量存在被发现以来，它们的重要生理机能及疾病标志物作用已逐渐成为作用于神经及内分泌系统的新型药物开发及新诊断方法开发的研究热点。然而，因体内大量存在的L-型氨基酸和内源性干扰物的影响，体内样品中酸性氨基酸D，L体的高选择性同时快速定量分析一直难以实现，其生理作用也难以进一步阐明。本研究旨在申请人过去的2D-HPLC研究基础上，开发新型高效整体手性色谱柱及ODS柱，并配合多样品环柱切换技术建立高选择性的迅速手性拆分的2D-HPLC全自动化系统，以实现哺乳动物组织和体液中微量的D-型酸性氨基酸及其L-异构体的高专属性同时测定。并应用该系统研究D-天门冬氨酸氧化酶基因敲除型鼠和多种疾病模型鼠体内组织及体液中D及L型酸性氨基酸的分布，为揭示其在神经，内分泌及免疫系统中的生理药理作用及疾病诊断标志物作用；开发如D-型氨基酸类保健食品和DDO抑制剂等药物提供科学依据。

自 D-型酸性氨基酸在哺乳动物体内的微量存在被发现以来，它们的重要生理机能及疾病标志物作用已逐渐成为作用于神经及内分泌系统的新型药物开发及新诊断方法开发的研究热点。本项目的主要研究内容是开发新型高效整体手性色谱柱，建立高选择性的迅速手性拆分的2D-HPLC系统，以实现哺乳动物组织和体液中微量的D-型酸性氨基酸及其L-异构体的高专属性同时测定，并应用该系统研究疾病模型鼠体内组织及体液中D 及L 型酸性氨基酸的分布。.经过本项目为期三年的研究，取得如下重要结果.1. 用双氢奎尼丁为（QD）为手性单体，开发出了用于快速手性分离酸性氨基酸对映体的键合型手性整体柱。对制备工艺进行了详细的筛选和优化，所制备出的QD手性整体柱对多种氨基酸异构体有较快的分析速度和较好的分离度。应用到小肽样品的分离中，实现了药物制剂中的小肽定量。.2. 建立了二维手性高效液相色谱自动分析系统。本系统实现了对生物样品中酸性氨基酸异构体的高效快速分离（Rs>2.5）；高灵敏度检测（LOD = 1 fmol）及定量值的确证。并首次发现了免疫不全小鼠（SAMP1）模型胸腺和脾脏中D-天门冬氨酸的含量较对照组同龄抗快速老化小鼠亚系1（SAMR1）有显著升高趋势（P<0.01），揭示出D-氨基酸有可能成为免疫不全性疾病的标志物。.3. 分析了DDO基因敲除程度和体内D型氨基酸含量之间的关系，发现了在DDO基因敲除小鼠中D-天门冬氨酸的急剧升高趋势。为开发如D-型氨基酸类保健食品和DDO 抑制剂等药物提供科学依据。.4. 首次成功实现了手性整体柱与荧光检测器的联用，克服了以往氨基酸异构体经手性整体柱分离后无法高灵敏度检测的问题，为手性整体柱在实际样品中的手性氨基酸分离中的应用奠定了基础。