采用化学发光-高效液相色谱法测定人血浆中的磷脂氢过氧化物。

Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescence-HPLC assay.

作者信息

Miyazawa T

机构信息

Department of Food Chemistry, Tohoku University, Sendai, Japan.

出版信息

Free Radic Biol Med. 1989;7(2):209-17. doi: 10.1016/0891-5849(89)90017-8.

DOI:10.1016/0891-5849(89)90017-8

PMID:2806945

Abstract

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was developed (Miyazawa, T. et al., Anal. Lett., 20, 915-925, 1987) and applied for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in biological tissues such as human blood plasma (Miyazawa, T. et al., Anal Lett 21:1033-1044, 1988; J. Biochem. 103:744-746; 1988). This system involves separation of phosphatidylcholines from plasma total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence of PCOOH. The chemiluminescence is produced by luminol oxidation during a reaction of hydroperoxide and cytochrome c-heme. The high specificity for hydroperoxide base enables a sensitive assay for a large range of PCOOH, with the detection limit of 10 picomole of hydroperoxide-O2. By use of this assay system, the presence of PCOOH in human blood plasma is confirmed quantitatively. The PCOOH concentration of healthy plasma is in the range below 10 nM to 500 nM, and much higher concentrations (500-9000 nM) of PCOOH are observed in the plasma of unhealthy donors.

摘要

开发了一种化学发光-高效液相色谱（CL-HPLC）系统（宫泽，T.等人，《分析快报》，20，915 - 925，1987），并将其应用于生物组织如人血浆中磷脂酰胆碱氢过氧化物（PCOOH）的氢过氧化物特异性测定（宫泽，T.等人，《分析快报》21:1033 - 1044，1988；《生物化学杂志》103:744 - 746；1988）。该系统包括用正相硅胶HPLC从血浆总脂质中分离磷脂酰胆碱，并对PCOOH的氢过氧化物依赖性化学发光进行柱后检测。化学发光是在氢过氧化物与细胞色素c-血红素反应过程中鲁米诺氧化产生的。对氢过氧化物碱基的高特异性使得能够对大范围的PCOOH进行灵敏测定，氢过氧化物-O2的检测限为10皮摩尔。通过使用该检测系统，定量证实了人血浆中PCOOH的存在。健康血浆中PCOOH的浓度在10 nM至500 nM以下的范围内，而在不健康供体的血浆中观察到更高浓度（500 - 9000 nM）的PCOOH。