Okimoto Yuko, Warabi Eiji, Wada Youichiro, Niki Etsuto, Kodama Tatsuhiko, Noguchi Noriko
Research Center for Advanced Science and Technology, University of Tokyo, Meguro, Tokyo, Japan.
Free Radic Biol Med. 2003 Sep 15;35(6):576-85. doi: 10.1016/s0891-5849(03)00330-7.
Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxide stoichiometrically to yield a fluorescent product DPPP oxide (DPPP=O) and the corresponding hydroxide, was used as a fluorescent probe for lipid peroxidation in low-density lipoprotein (LDL). DPPP was successfully incorporated into LDL using the dispersion reagent Pluronic F-127. Incorporation of DPPP into LDL was confirmed by gel filtration chromatography. Reaction of DPPP with hydroperoxide within an LDL particle was examined by monitoring the increase in fluorescence intensity of the LDL. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably reacted with DPPP, whereas hydrogen peroxide did not. Fluorescence was increased at the early stages in the oxidation of DPPP-labeled LDL by an azo radical initiator or human neutrophils. LDL, which was labeled with DPPP or DPPP=O, was taken up by cells such as THP-1-derived macrophages and human umbilical vein endothelial cells. The fluorescence of DPPP=O could be observed in cells using fluorescence microscopy equipped with a cooled charge coupled device camera in a nondestructive manner. The present study shows that DPPP is a sensitive, selective, and quantitative probe for monitoring LDL oxidation and visualizing intracellular oxidation.
二苯基-1-芘基膦（DPPP）与脂质氢过氧化物按化学计量反应生成荧光产物二苯基-1-芘基膦氧化物（DPPP=O）和相应的氢氧化物，被用作低密度脂蛋白（LDL）中脂质过氧化的荧光探针。使用分散剂普朗尼克F-127成功地将DPPP掺入LDL中。通过凝胶过滤色谱法确认了DPPP掺入LDL中。通过监测LDL荧光强度的增加来研究DPPP与LDL颗粒内氢过氧化物的反应。发现脂溶性氢过氧化物如亚油酸甲酯氢过氧化物优先与DPPP反应，而过氧化氢则不反应。在用偶氮自由基引发剂或人中性粒细胞氧化DPPP标记的LDL的早期阶段，荧光增强。用DPPP或DPPP=O标记的LDL被THP-1衍生的巨噬细胞和人脐静脉内皮细胞等细胞摄取。使用配备有冷却电荷耦合器件相机的荧光显微镜可以无损地在细胞中观察到DPPP=O的荧光。本研究表明，DPPP是一种用于监测LDL氧化和可视化细胞内氧化的灵敏、选择性和定量的探针。
