Development of HPLC-Fluorescence Determination method of NO and Its Application to Biological Samples

HPLC-荧光测定生物样品NO方法的建立及其应用

• 批准号: 09672189
• 负责人: NAKASHIMA Kenichiro
• 金额: $ 1.86万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672189/
• 关键词: nitric oxide; high-performance liquid chromatography; 2,3-diaminonaphtalene; hemoglobins; 1(H)-2,3-naphthotriazole; cultivated plant cells; NO-production; 蛍光検出; 植物培養細胞

A high-performance liquid cliromatographic (HIPLC) determination of NO with fluorescence reaction product was confirmed The fluorescent reaction product was confirmed to be 1t(H)-2,3-naphthotriazole (NT) by an instrumental analysis. The peak on the chromatogram dcrived from the reaction product of SNAP with 2,3-diaminonaphthalene (DAN) was well corresponded to that of NT.Trapping abilities of some hemoglobins for NO were evaluated by using the developed method The order of trapping abilities of these compounds including dimethyldithiocarbamic acid (DDCT)-Fe complex, was DDTC-Fe >> oxyhemoglobin*myoglobin > hemoglobin >> methehemoglobin.The present method was applied to the determination of NO in a cultured plant cells. After the incubation of cultured cells of Agave pacittica with DAN for 3h, cells were homogenized in methanol and centrifuged. Portions of 20 muI of the supernatant were injected onto HPLC.The detection limit of standard NO-DAN was 8.0 fmol on column (S/N=3). The incorporation rate of DAN into plant cells was about 19.6%, and the estimated NO concentration in cells was 5.3*1.6 pmol/g (n=3). A study on the role of NO in plant tissues is under investigation.

荧光反应产物的高效液相色谱(HIPLC)测定NO被证实。通过仪器分析，荧光反应产物证实为1t(H)-2,3-萘并三唑(NT)。 SNAP 与 2,3-二氨基萘 (DAN) 反应产物的色谱峰与 NT 的峰非常一致。使用开发的方法评估了一些血红蛋白对 NO 的捕获能力。这些化合物（包括二甲基二硫代氨基甲酸 (DDCT)-Fe 络合物）的捕获能力顺序为 DDTC-Fe >> 氧合血红蛋白*肌红蛋白>血红蛋白>>高铁血红蛋白。本方法适用于培养植物细胞中NO的测定。将龙舌兰培养细胞与DAN一起孵育3小时后，将细胞在甲醇中匀浆并离心。取20μL上清液注入HPLC。标准NO-DAN的检出限为8.0fmol(S/N=3)。 DAN进入植物细胞的掺入率约为19.6%，估计细胞内NO浓度为5.3*1.6 pmol/g（n=3）。关于一氧化氮在植物组织中的作用的研究正在进行中。

项目成果

Yining Zhao: "Chemiluminescence Determination of Tiopronin by Flow Injection Analysis Based on Cerium(IV)---" Analyst. 112. 103-106 (1997)

赵一宁：“基于铈(IV)的流动注射化学发光测定硫普罗宁——”分析师。

Yining Zhao: "Chemiluminescence Determination of Tiopronin by Flow Injection Analysis Based on Cerium(IV) Oxidation Sensitized by Quinine" Analyst. 112. 103-106 (1997)

赵一宁：“基于奎宁敏化的铈（IV）氧化的流动注射分析化学发光测定硫普罗宁”分析师。

Yining Zhao: "Chemiluminescence Determination of Tiopronin and its Metabolite 2-Mercaptopropionic Acid---" Chromatographia. 44. 31-36 (1997)

赵一宁：“硫普罗宁及其代谢物2-巯基丙酸的化学发光测定——”色谱法。

Yining Zhao: "Chemiluminescence Determination of Tiopronin by Flow Injection Analysis Based on Cerium (IV) Oxidation ---" Analyst. 122. 103-106 (1997)

赵一宁：“基于铈（IV）氧化的流动注射化学发光测定硫普罗宁---”分析师。

Mitsuhiro Wada: "A Simple and Selective Monitoring Method for Nitric Oxide Capturing Ability by HPLC-----" Analytical Sciences. 14. 1177-1179 (1998)

Mitsuhiro Wada：“通过 HPLC 检测一氧化氮捕获能力的简单且选择性的监测方法 -----”分析科学。