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

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a f luorescent probe for lipid peroxidation in live cells. DPPP was successfull y incorporated into U937 cells. Incorporation of DPPP into the cell membran e was confirmed by fluorescence microscopy. Reaction of DPPP with hydropero xides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linolea te hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between inc rease in fluorescence intensity and the amount of methyl linoleate hydroper oxide applied to the cell was observed. DPPP cave little effect on cell pro liferation, cell viability or cell morphology for at least 3 d. DPPP oxide. fluorescent product of DPPP, was quite stable in the membrane of living ce lls for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2'-Azobis(2-amidinopropane) dihyd rochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluoresce nce. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H2 O2, 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 thi s probe is especially advantageous in long-term peroxidation of the cell. ( C) 2001 Elsevier Science Inc.