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High-Sensitive Analysis of the Free-Radical Scavenging Products of Vitamin E in Biological Samples by High-Performance Liquid Chromatography

采用高效液相色谱法高灵敏度分析生物样品中维生素 E 的自由基清除产物

基本信息

批准号：
11660124
负责人：
YAMAUCHI Ryo
金额：
$ 2.3万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660124/
关键词：
Vitamin E α-Tocopherol Lipid peroxidation Cholesterol ester Phosphatidylcholine HPLC Free radical 化学発光検出

项目摘要

A simple and sensitive method using a high-performance liquid chromatography (HPLC) system with post-column reduction using platinum or zinc as the catalyst followed by electrochemical detection (ECD) has been developed for the quantification of the oxidation products of vitamin E (α-tocopherol, α-TH), including 8a-(phosphatidylcholine-dioxy)-α-tocopherones (TOO-PCs), 8a-(cholestrylester-dioxy)-α-tocopherones (TOO-ChEs), α-tocopherylquinone (TQ), and epoxy-α-tocopherylquinones (epoxyTQs). The lipids extracted from biological samples were treated with solid-phase extraction of a silica-NH_2 cartridge before the HPLC analysis ; THs, TQ, epoxyTQs, and TOO-ChEs were eluted with chloroform, TOO-PCs being with methanol, respectively. THs, TQ, and epoxyTQs in the chloroform fraction were separated by a J'sphere ODS-H80 column with 95% methanol containing 10 mM lithium acetate and 5 mM trifluoroacetic acid and quantified by ECD after on-line zinc-catalyzed reduction. TOO-PCs in the methanol fr … More action were separated by a J'sphere ODS-L80 column using 97.5% methanol containing 20 mM choline chloride, passed through a reduction column (5% platinum on alumina), and the reduced forms were quantified by ECD (+700 mV) ; TOO-ChEs in the chloroform fraction being the same conditions with 98.5% methanol containing 10 mM lithium acetate as the solvent. The detection limit for each compound by this method was about 0.2 pmol. This method was applied to the detection of the reaction products of a-TH in the peroxidized human plasma. The endogenous α-TH in plasma could scavenge peroxyl radicals of cholesterylester (ChE) and phosphatidylcholine (PC) to suppress the formation of ChE and PC hydroperoxides when the peroxidation was initiated by a free radical initiator at 37℃. The main products detected by reversed-phase HPLC were the addition products of TH with ChE-peroxyl radicals (TOO-ChE), TQ, and epoxyTQ.The structures of TOO-ChE were characterized as cholestryl (8a-dioxy-α-tocopherone)-epoxyoctadecenoates. The results indicate that endogenous α-TH in human plasma can react with lipid peroxyl radicals to form some reaction products.In conclusion, the HPLC technique presented here is found to advantages for the sensitive and specific determination of the free-radical scavenging products of vitamin E in biological samples. Less

开发了一种简单而灵敏的方法，使用高效液相色谱 (HPLC) 系统，使用铂或锌作为催化剂进行柱后还原，然后进行电化学检测 (ECD)，用于定量维生素 E (α-生育酚，α-TH) 的氧化产物，包括 8a-(磷脂酰胆碱-二氧基)-α-生育酚 (TOO-PC)、8a-(胆固醇酯-二氧基)-α-生育酚 (TOO-ChE)、α-生育醌 (TQ) 和环氧-α-生育醌 (epoxyTQ)。从生物样品中提取的脂质在进行HPLC分析之前先用硅胶-NH_2小柱进行固相萃取处理； THs、TQ、epoxyTQs 和 TOO-ChEs 分别用氯仿洗脱，TOO-PCs 用甲醇洗脱。氯仿馏分中的 TH、TQ 和环氧 TQ 通过 J'sphere ODS-H80 柱使用含有 10 mM 乙酸锂和 5 mM 三氟乙酸的 95% 甲醇进行分离，并在在线锌催化还原后通过 ECD 进行定量。甲醇中的 TOO-PC 通过 J'sphere ODS-L80 柱分离，使用含有 20 mM 氯化胆碱的 97.5% 甲醇，通过还原柱（氧化铝上的 5% 铂），并通过 ECD (+700 mV) 对还原形式进行定量；氯仿馏分中的 TOO-ChEs 的条件相同，以含有 10 mM 醋酸锂的 98.5% 甲醇为溶剂。该方法对每种化合物的检测限约为 0.2 pmol。该方法适用于过氧化人血浆中α-TH反应产物的检测。当自由基引发剂在37℃引发过氧化反应时，血浆中的内源性α-TH可以清除胆固醇酯(ChE)和磷脂酰胆碱(PC)的过氧自由基，从而抑制ChE和PC氢过氧化物的形成。反相HPLC检测到的主要产物为TH与ChE-过氧化自由基的加成产物(TOO-ChE)、TQ和环氧TQ。TOO-ChE的结构经表征为胆甾醇(8a-二氧基-α-生育酮)-环氧十八烯酸酯。结果表明，人血浆中的内源性α-TH可以与脂质过氧化自由基反应，形成一些反应产物。总之，本文提出的HPLC技术对于生物样品中维生素E自由基清除产物的灵敏和特异测定具有优势。较少的