Development of Specific and Sensitive Fluorogenic Reagents for Bioactive Amines and Their Applications

本课题利用研制的几种生物活性胺的特异荧光试剂，建立了几种生物活性胺的特异、灵敏、快速测定方法。以1,2 -二苯甲酰- 1,4 -二氢苯为柱前荧光标记试剂，建立了高效液相色谱法测定生物样品中腐胺的方法。该方法适用于大鼠肝脏中鸟氨酸脱羧酶（ODC）的测定。采用组胺特异性荧光试剂4-溴- 1,2 -苯二醛（BBDA），建立了一种测定生物样品中组胺的简单荧光法。该试剂适用于流动注射分析（FIA）方法，可在1小时内检测40个样品。该方法对组胺的检出限（S/N=5）为0.5 pmo1/支。以2-乙酰苯甲醛（ABA）为柱后荧光标记试剂，建立了高效液相色谱法测定组胺的方法。该方法灵敏度高，适用于组织提取物脱蛋白后的组胺含量测定。在适当的反应条件下，用ABA可作为谷胱甘肽的特异试剂。因此，该试剂可用于简单荧光法和快速FIA法测定谷胱甘肽。以2,3 -萘二乙醛为精氨酸特异性荧光试剂，建立了灵敏、快速的血清精氨酸荧光定量分析方法。此外，发现2,5 -二甲氧基四氢呋喃是色氨酸的特异性荧光试剂，适用于氨基酸混合物中色氨酸的荧光测定方法。

In this research project, specific, sensitive and rapid methods were established for the assay of several bioactive amines by using their specific fluorogenic reagents we had developed.1. High-performance liquid chromatographic(HPLC) method was developed for the determination of putrescine in biological samples by using 1, 2-dibenzoyl-1, 4-dihydrobenzene as a precolumn fluores-cence labelling reagent. This HPLC method was shown to be applicable to the assay of ornithine decarboxylase(ODC) in rat livers.2. By using 4-bromo-1, 2-benzenedicarbaldehyde(BBDA), a specific fluorogenic reagent for histamine, we developed a simple fluorometric method for the determination of histamine in biological samples. This reagent was applied to the flow injection analysis(FIA) method, which allows 40 samples to be assayed within 1 hour. The detection limit(S/N=5) of this FIA method for histamine was demonstrated to be 0.5 pmo1/injection.HPLC method for assay of histamine was also developed by using 2-acetylbenzaldehyde(ABA) as a postcolumn fluorescence labelling reagent. This method was highly sensitive and applicable to the assay of histamine in tissue extract only after deproteinization.3. Under the appropriate reaction conditions used ABA could be employed as a specific reagent for glutathione. Thus, this reagent was employed to the simple fluorometric method and the rapid FIA method for the assay of glutathione.4. By using 2, 3-naphthalenedicarbaldehyde, a specific fluorogenic reagent for arginine, sensitive and rapid FIA method was developed for the assay of arginine in serum. Furthermore, 2, 5-dimethoxytetrahydrofuran was found to be specific fluorogenic reagent for tryptophane and applicable to the fluorometric method for the assay of tryptophane in amino acid mixtures.