Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide
Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide
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DOI：
10.1007/s11745-005-1412-2
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发表时间：
2005-05-01
期刊：
影响因子：
1.9
通讯作者：
Kurosawa, T
中科院分区：
文献类型：
--
作者：
Hui, SP;Murai, T;Kurosawa, T
我们建立了一种灵敏的反相化学发光高效液相色谱法，用于同时定量和定性分析人血浆中胆固醇酯和甘油三酯的氢过氧化物。胆固醇酯和TG的标准氢过氧化物和新的内标物（1 -十四烷基3-十八烯酰氧基-5 β-胆烷-24-酸酯单氢过氧化物）（I. S.）化学合成，并证实标准曲线在整个校准范围（1-1000 pmol）内呈线性。日内、日间变异系数均小于7%，回收率在84- 93%之间。每次运行的样本量最小化至0.1 mL血浆，健康年轻人（22-25岁; n = 15，男性/女性= 6：9）的血浆胆固醇酯氢过氧化物水平为189 +/- 87 nM（平均SD），健康老年人（39-60岁; n = 6，男性/女性= 3：3）的血浆胆固醇酯氢过氧化物水平为210 69 nM。TG氢过氧化物在健康受试者中未检测到。在晚期肝衰竭患者（36-67岁; n = 4，男性/女性= 2：2）中，血浆胆固醇酯和TG的氢过氧化物水平分别为11，903 - 9，553 nM和3，318 - 1，590 nM，表明脂质氧化的参与。使用本发明的化学发光HPLC方法，用合成的I. S.可能有助于我们理解脂质过氧化的化学和病理生理学方面。
We have developed a sensitive reversed-phase chemiluminescence HPLC approach for simultaneous quantitative and qualitative analyses of hydroperoxides of cholesteryl ester and TG in human plasma. Standard hydroperoxides of cholesteryl ester and TG and a novel internal standard (1 -tetradecanyl 3-octadecenoyloxy-5 beta-cholan-24-oate monohydroperoxicle) (I.S.) were chemically synthesized and the standard curves confirmed to be linear throughout the calibration range (1-1000 pmol). Within-day and between-day CV were less than 7%, and the recoveries were within the range of 84-93%. With sample size minimized to 0.1 mL of plasma for each run, plasma cholesteryl ester hydroperoxide levels were 189 +/- 87 nM (mean SD) in healthy young (22-25 yr old; n = 15, male/female = 6:9) and 210 69 nM in healthy elderly (39-60 yr old; n = 6, male/female = 3:3). TG hydroperoxide was not detected in healthy subjects. In patients with advanced liver failure (36-67 yr old; n = 4, male/female = 2:2), hydroperoxide levels of plasma cholesteryl ester and TG were 11,903 9,553 nM and 3,318 1,590 nM, respectively, indicating an involvement of lipid oxidation. Sensitive and specific monitoring of plasma lipid peroxides using the present chemiluminescence HPLC approach with the synthesized I.S. may help our understanding of chemical and pathophysiological aspects of lipid peroxidation.