A novel method of following oxidation of low-density lipoprotein using a sensitive fluorescent probe, diphenyl-1-pyrenylphosphine.
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.
Okimoto
Yuko
Y
Research Center for Advanced Science and Technology, University of Tokyo, Meguro, Tokyo, Japan.
Warabi
Eiji
E
Wada
Youichiro
Y
Niki
Etsuto
E
Kodama
Tatsuhiko
T
Noguchi
Noriko
N
D002460:Cell Line; D002474:Cell-Free System; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes / Q000032:analysis; D015394:Molecular Structure; D009504:Neutrophils; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D011721:Pyrenes / Q000737:chemistry*
D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D015335:Molecular Probes; D009943:Organophosphorus Compounds; D011721:Pyrenes; C066322:diphenyl-1-pyrenylphosphine