Development of Peroxidized Lipid Detection Method using TLC Blot

建立了一种特异性检测脂质过氧化氢的薄层色谱印迹技术。通过将板浸入含有二苯基-1-芘膦（DPPP）的印迹溶剂中，将通过与大豆脂氧合酶反应制备的范围为0.2至0.5 nmol的磷脂酰胆碱氢过氧化物（PC-OOH）和胆固醇亚油酸氢过氧化物（CE-OOH）在印迹膜上显现为荧光点。该技术已成功地应用于体外人低密度脂蛋白（LDL）脂质过氧化反应的监测。我们应用这种方法从生物样品中方便地分离LOOH及其衍生为羟基化合物。作为第一步，通过TLC印迹和GC-MS的组合进行人LDL中CE-OOH的分析，因为氧化LDL已被暗示为动脉粥样硬化发展的可能生物标志物。用铜离子（CuS 〇 4）（水溶性偶氮自由基产生剂（AAPH））氧化人LDL， ...更多信息 光敏剂+光或15-脂氧合酶（15-LOX）。脂质提取后，将样品用含有DPPP的溶剂进行TLC印迹。去除标准品CE-OOH的荧光点，提取CE-OOH的羟基衍生物，再进行氢化、甲基转移和三甲基硅烷化衍生，进行GC-MS/SIM分析。通过这些程序，异构体CE-OOH积累在LDL中被确定为甲基羟基十八烷酸酯的三甲基甲硅烷基衍生物。在每种情况下，我们都成功地测定了LDL氧化产生的同分异构体CE-OOH。这里描述的方法作为一个方便和灵敏的技术，用于测定异构体LOOH从生物样品。在应用抗氧化活性的情况下，通过TLC印迹技术的分析表明，胃粘膜具有最高的消除PC-OOH的潜力。此外，在印迹膜中检测到的游离脂肪酸氢过氧化物的事实强烈地表明磷脂酶A2和GSH-过氧化物酶参与胃粘膜对PC-OOH的解毒。少

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