DEVELOPMENT OF HIGHLY SELECTIVE AND SENSITIVE DETERMINATION METHOD FOR LIPID PEROXIDES AND ITS APPLICATION TO BIOLOGICAL SAMPLES

脂质过氧化物高选择性、灵敏测定方法的建立及其在生物样品中的应用

• 批准号: 16590030
• 负责人: KURODA Naotaka
• 金额: $ 2.24万
• 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: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590030/
• 关键词: Lipid peroxide; UV irradiation; Chemiluminescence detection; Hydroperoxide; Fenton reaction; 科学発光検出

Highly selective and sensitive determination of lipid peroxides was examined by the use of chemiluminescence (CL) detection. For this purpose, a high-performance liquid chromatographic (HPLC) system with on-line photoreactor and peroxyoxalate chemiluminescence (PO-CL) detection was developed. The PO-CL reaction requires hydrogen peroxide, fluorophore and diaryloxalate in order to show CL. In the proposed system, hydrogen peroxide is generated from lipid peroxide under UV radiation, and thus only the lipid peroxide labeled with fluorophore is expected to show CL, which makes the method very selective.The system was evaluated by using standard lipid peroxides, which were prepared by photosensitized oxidation of oleic acid in the presence of methylene blue and followed by fluorescence (FL) labeling with 4,5-diphenyl-2-(4-hydrazinocarbonyl)phenyl imidazole (HCPI) or 2-(2,3-naphthalimino)ethyltrifluoromethane sulfonate (NE-OTf). As a result, the method was found to be selective for lipid peroxides but was not as sensitive as expected. This was due to the low generation yield of hydrogen peroxide from hydroperoxide by UV irradiation. We therefore attempted to increase the sensitivity by using Fenton reaction instead of UV irradiation. Fifteen-fold increase in CL intensity was observed by using the Fenton reaction. This sensitivity will further be improved by optimizing the Fenton reaction.In this research, we selected the analyte molecule having hydroperoxide and fluorophore for PO-CL detection. However, the fluorophore will be able to replace with chemiluminophore. The sensitivity of the method will be improved by examining the reaction that generates reactive oxygen species from lipid peroxides for the efficient CL reaction.

采用化学发光检测法对脂质过氧化物进行了高选择性、高灵敏度的测定。为此，建立了一种高效液相色谱（HPLC）在线光反应器和过氧草酸盐化学发光（PO-CL）检测系统。PO-CL反应需要过氧化氢，荧光团和二芳基草酸酯以显示CL。该体系中，过氧化脂质在紫外光照射下产生过氧化氢，因此只有荧光标记的过氧化脂质才能显示出CL，这使得该方法具有很高的选择性。该体系通过使用标准过氧化脂质进行评价，标准过氧化脂质是通过在亚甲基蓝存在下光敏氧化油酸制备的，然后用4，5-二苯基-2-（4-肼基羰基）苯基咪唑（HCPI）或2-（2，3-萘亚氨基）乙基三氟甲磺酸酯（NE-OTf）。因此，发现该方法对脂质过氧化物具有选择性，但不如预期灵敏。这是由于通过UV照射从氢过氧化物生成过氧化氢的产率低。因此，我们试图通过使用芬顿反应代替紫外照射来提高灵敏度。通过使用芬顿反应，观察到CL强度增加了15倍。通过优化芬顿反应体系，可以进一步提高检测灵敏度。然而，荧光团将能够被荧光团取代。该方法的灵敏度将通过检查从脂质过氧化物中产生活性氧的反应来提高，以实现有效的CL反应。

项目成果

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction

使用合成底物作为鲁米诺化学发光反应的增强剂进行脂肪酶活性的化学发光测定

• 发表时间: 2004
• 期刊: Luminescence 19
• 作者: T.Ichibangase;et al.
• 通讯作者: et al.

Capillary electrochromatography of caffeine and its metabolites in rat brain using microdialysis technique

使用微透析技术对大鼠脑内咖啡因及其代谢物进行毛细管电色谱分析

• 发表时间: 2005
• 期刊: Electrophoresis 26
• 作者: K.Ohyama;et al.
• 通讯作者: et al.

Retrospective analyses of atmospheric polycyclic and nitropolycyclic aromatic hydrocarbons in an industrial area of a western site of Japan

日本西部工业区大气中多环芳烃和硝基多环芳烃的回顾分析

• 发表时间: 2005
• 期刊: Analytical Sciences 21
• 作者: Naoya Kishikawa;et al.
• 通讯作者: et al.

抗酸化物質の次亜塩素酸イオン消去能測定のためのシーケンシャルインジェクション-ルミノール化学発光分析法の開発

连续注射鲁米诺化学发光光谱法测定抗氧化剂次氯酸根离子清除能力的开发

• 发表时间: 2004
• 期刊: 分析化学 53
• 作者: Ito;T.;中村 訓子ら
• 通讯作者: 中村 訓子ら

Capillary electrochromatography of caffeine and its metabolites in rat brain microdialysate.

大鼠脑微透析液中咖啡因及其代谢物的毛细管电色谱法。

• 发表时间: 2005
• 期刊: Electrophoresis 26
• 作者: Naotaka Kuroda;et al.
• 通讯作者: et al.