Estimation of lipid peroxidation of live cells using a fluorescent probe, Diphenyl-1-pyrenylphosphine

Abstract

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a fluorescent probe for lipid peroxidation in live cells. DPPP was successfully incorporated into U937 cells. Incorporation of DPPP into the cell membrane was confirmed by fluorescence microscopy. Reaction of DPPP with hydroperoxides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between increase in fluorescence intensity and the amount of methyl linoleate hydroperoxide applied to the cell was observed. DPPP gave little effect on cell proliferation, cell viability or cell morphology for at least 3 d. DPPP oxide, fluorescent product of DPPP, was quite stable in the membrane of living cells for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluorescence. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H 2O 2, AAPH, and DEM to compare the formation of hydoperoxides in the membrane and cytosol, distinct patterns of peroxide formation were observed. These results indicate that fluorescent probe DPPP is eligible for estimation of lipid peroxidation proceeding in the membrane of live cells, and use of this probe is especially advantageous in long-term peroxidation of the cell.