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.

建立了高效液相色谱(HIPLC)结合荧光反应产物测定NO的方法。仪器分析结果表明，荧光反应产物为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峰有很好的对应关系。用建立的方法评价了几种血红蛋白对NO的捕集能力。这些化合物包括二甲基二硫代氨基甲酸(DDCT)-铁的络合物对NO的捕集能力大小顺序为DDTC-Fe&gt；&gt；氧合血红蛋白*肌红蛋白&gt；血红蛋白&gt；&gt；高铁血红蛋白。将培养的龙舌兰细胞与DAN孵育3h后，在甲醇中匀浆，离心。将20微升的上清液进样到高效液相色谱仪上，柱上标准亚硝酸根的检出限为8.0fmol(S/N=3)。DAN对植物细胞的参入率约为19.6%，细胞内NO浓度估计为5.3×1.6pmol/g(n=3)。一项关于NO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。