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

高效液相色谱/化学发光测定生物硫醇的开发及应用

• 批准号: 63571023
• 负责人: NAKASHIMA Kenichiro
• 金额: $ 1.28万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571023/
• 关键词: Biological thiols; High-performance liquid chromatography; Chemiluminescence detection; Aryl oxalate; Lipoperoxide; Malondialdehyde; Peroxyoxalate chemiluminescence; 生体チオ-ル; 生体チオール; 化学発光; 高速液体クロマトグラフィー; アリールオキザレート; ピリミドピリミジン誘導体

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.

本研究取得的结果如下： 1．建立了一种以N-[4-(6-二甲氨基-2-苯并呋喃基)苯基]马来酰亚胺(DBPM)为荧光标记的高效液相色谱(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。该方法非常灵敏，可以检测 fmol 硫醇水平。接下来，开发了另一种使用 4-氟-7-硝基苯并-2,1,3-恶二唑 (NBD-F) 作为标记的 HPLC-CL 方法。但该方法的灵敏度较低，为pmol水平。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。可能是所用化学发光试剂不合适造成的。2．为了评估草酸盐和荧光剂作为过草酸盐 CL 试剂，我们开发了一种简单快速的流动系统方法。通过该方法，对新合成的草酸芳基酯和荧光化合物进行了评价，发现了一些可用作化学发光试剂的化合物。该方法应适合HPLC-过草酸CL试剂的选择。 3．采用方法2测定丙二醛(MDA)与硫代巴比妥酸缩合物的CL效率。结果，MDA-1,3-二乙基-2-硫代巴比妥酸 (DETBA) 缩合物表现出最大的 CL 效率，并且可以检测到低至 10 fmol 的浓度。这表明 DETBA 将是一种极好的 MDA 衍生化试剂。通过使用 DETBA，应开发一种高灵敏度的过氧化脂质 (MDA) 测定方法。

项目成果

R. S. Givens: Peroxyoxalate Chemiluminescence Reaction: A Versatile Method for Activation of Fluorophore Luminescence. ACS Symposium Series 383, Chapter 8, 127-154, 1989,

R. S. Gives：过氧草酸化学发光反应：一种激活荧光团发光的多功能方法。

W.R.G.Baeyens: "Development of Chemiluminescence in Biomedical Analysis" J.Pharm.Biomed.Anal.7. 407-412 (1989)

W.R.G.Baeyens：“化学发光在生物医学分析中的发展”J.Pharm.Biomed.Anal.7。

中島憲一郎: "生物発光と化学発光" 廣川書店, 77-108 (1989)

中岛健一郎：“生物发光和化学发光”广川书店，77-108（1989）

Kenichiro Nakashima: "Synthesis of Pyrimido(5、4-d)pyrimidine Derivatives and Their Ultraviolet Absorption and Fluorescence Spectral Properties" Dyes and Pigments. 12. 21-26 (1990)

Kenichiro Nakashima：“嘧啶并(5,4-d)嘧啶衍生物的合成及其紫外线吸收和荧光光谱特性”染料和颜料12. 21-26 (1990)。

K. Nakashima: "High-performance Liquid Chromatography/Chemiluminescence Determination of Biological Thiols with N-[4-(6-Dimethylamino-2-benzofuranyl)phenyl]maleimide" Biomed. Chromatogr., 3(1), 39-42, 1989.

K. Nakashima：“用 N-[4-(6-二甲氨基-2-苯并呋喃基)苯基]马来酰亚胺进行生物硫醇的高效液相色谱/化学发光测定”Biomed。