Development of Peroxidized Lipid Detection Method using TLC Blot

开发使用 TLC 印迹检测过氧化脂质的方法

• 批准号: 12556020
• 负责人: TERAO Junji
• 金额: $ 1.98万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556020/
• 关键词: peroxidized lipids; TLC blot; atherosclerosis; denatured LDL; lipid hydroperoxides; plasma lipids; cholesteryl ester; phospholipids

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides (PC-OOH) and cholesteryl linoleate hydroperoxides (CE-OOH) ranging from 0.2 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescence spots on the blotted membranes by immersing the plate into a blotting solvent containing diphenyl-1-pyrenyphosphine (DPPP). This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein (LDL) in vitro. We applied this methodology for convenient isolation of LOOH from biological samples and its derivatization to hydroxyl compound. As a first step, analysis of CE-OOH in human LDL was performed by the combination of TLC blotting and GC-MS, because oxidized LDL has been implied as a possible biomarker for the development of atherosclerosis. Human LDL was oxidized with copper ion (CuS04), a water-soluble azo radical generator (AAPH) … More , photosensitizer +light, or 15-lipoxygenase (15-LOX). After lipid extraction, samples were applied to TLC blotting with the solvent containing DPPP. The fluorescent spot corresponding to standard CE-OOH was removed and extracted as hydroxyl derivative of CE-OOH Then the samples were subjected to GC-MS/SIM analysis after additional derivatization involving hydrogenation, transmethylation and trimethylsilylation. By these procedures, isomeric CE-OOH accumulated in the LDL were determined as trimethylsilyl derivatives of methyl hydroxyoctadecanoate. In each case, we succeeded in the determination of isomeric CE-OOH produced from the oxidation of LDL. The methodology described here serves as a convenient and sensitive technique for the determination of isomeric LOOH from biological samples. In the case of application for antioxidant activity, analysis by the TLC blot technique showed that the gastric mucosa has the highest potential to eliminate PC-OOH. Furthermore the fact that free fatty acid hydroperoxides detected in blotted membranes strongly suggested the participation of phospholipase A2 and GSH-peroxidase in the detoxification of PC-OOH by gastric mucosa. Less

建立了一种薄层色谱法特异性检测脂质氢过氧化物的印迹技术。用大豆脂氧合酶反应制备0.2 ~ 0.5 nmol范围内的磷脂酰胆碱氢过氧化物（PC-OOH）和胆固醇酰亚油酸氢过氧化物（CE-OOH），将板浸入含有二苯基-1-芘膦（DPPP）的印迹溶剂中，在印迹膜上可见荧光斑点。该技术成功地应用于体外监测人低密度脂蛋白（LDL）的脂质过氧化。我们应用该方法方便地从生物样品中分离LOOH并将其衍生为羟基化合物。作为第一步，我们通过TLC印迹和GC-MS相结合的方法对人LDL中的CE-OOH进行了分析，因为氧化LDL可能是动脉粥样硬化发展的生物标志物。用铜离子（CuS04）、水溶性偶氮自由基发生器（AAPH）、光敏剂+光或15-脂氧合酶（15-LOX）氧化人体LDL。脂质提取后，用含DPPP的溶剂进行TLC印迹。去除标准CE-OOH对应的荧光点，提取CE-OOH的羟基衍生物，再进行加氢、转甲基化和三甲基硅基化等衍生化处理，进行GC-MS/SIM分析。通过这些方法，LDL中积累的异构体CE-OOH被确定为羟十八烷酸甲酯的三甲基硅基衍生物。在每种情况下，我们都成功地测定了由LDL氧化产生的异构体CE-OOH。本方法为生物样品中同分异构体LOOH的测定提供了一种方便、灵敏的方法。在应用抗氧化活性的情况下，通过TLC blot技术分析表明，胃粘膜具有最大的消除PC-OOH的潜力。此外，在印迹膜中检测到的游离脂肪酸过氧化物强烈提示磷脂酶A2和gsh过氧化物酶参与了胃粘膜对PC-OOH的解毒作用。少

项目成果

Junji Terao: "Phospholipid Hydroperoxides Are Detoxified by Phospholipase A_2 and GSH Peroxidase in Rat Gastric Mucosa"Lipids. 38.6. 641-649 (2003)

Junji Terao：“磷脂酶 A_2 和 GSH 过氧化物酶对大鼠胃粘膜中的磷脂氢过氧化物进行解毒”脂质。

S.Miyamoto, K.Murota, J.Terao: "High Oxidizability of Large Intestinal Mucosa in Iron Ion-Induced Lipid Peroxidation in Oxidative Stress in Intestinal Mucosa"Basel, Karger. 10 (2001)

S.Miyamoto、K.Murota、J.Terao：“肠粘膜氧化应激中铁离子诱导的脂质过氧化作用中大肠粘膜的高氧化性”巴塞尔，Karger。

S.Miyamoto: "Phospholipid hydroperoxides are detoxified by Phospholipase A2 and Glutathione peroxidase in rat gastric mucosa"Lipids. (受理).

S.Miyamoto：“磷脂酶 A2 和谷胱甘肽过氧化物酶对大鼠胃粘膜中的磷脂氢过氧化物进行解毒”（已接受）。

Junji Terao: "Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides"Journal of chromatography B. 765. 199-203 (2001)

Junji Terao：“用于荧光检测磷脂氢过氧化物和胆固醇酯氢过氧化物的薄层色谱印迹”Journal of Chromatography B.765.199-203(2001)

Sayuri Miyamoto, Coralie Dupas, Kaeko Murota and Junji Terao: "Phospholipid hydroperoxides are detoxified by phospholipase A_2 and Glutathione peroxidase in rat gastric mucosa"Lipids. 38. 641-649 (2003)

Sayuri Miyamoto、Coralie Dupas、Kaeko Murota 和 Junji Terao：“大鼠胃粘膜中的磷脂酶 A_2 和谷胱甘肽过氧化物酶对磷脂氢过氧化物进行解毒”。