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, H₂O₂, 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.

Introduction

Generation of reactive oxygen species (ROS) and subsequent oxidative modification of biomolecules such as lipids, proteins, and nucleic acids is inevitable for aerobic organisms. While small amount of ROS has proved to be involved in important physiological functions such as signal transduction leading to gene expression and cell proliferation [1], [2], excess amount of ROS has been implicated in a variety of pathological events such as atherosclerosis [3], [4], ischemia-reperfusion injury [5], cardiovascular diseases [6], [7], and neurodegenerative diseases [8], [9]. Although detailed mechanisms by which ROS lead to these consequences are not fully understood, lipid peroxidation of the cell membrane has been considered to be critically involved. There are several ways for monitoring lipid peroxidation process, which include measurement of oxygen uptake, loss of lipid substrates such as polyunsaturated fatty acids, and accumulation of peroxidation products such as hydroperoxides and aldehydes.

A variety of methods have been developed for determination of the levels of lipid hydroperoxides or aldehydic end products such as malondialdehyde (MDA) in biological materials. Thiobarbituric acid reactive substances (TBARS) assay is the most popular biochemical analysis of peroxidation products, whereas quantification of lipid hydroperoxides using high performance liquid chromatography (HPLC) with chemiluminescence detection [10], [11] and fluorescence detection [12] proves to be most sensitive. Besides biochemical determination, noninvasive, real-time monitoring of lipid peroxidation using fluorescent probes has also been developed. Among several probes, cis-parinaric acid (PnA) has been the only practical one used in living cells [13], [14]. Common limitation of use of fluorescent probes, however, is that the probes often are cytotoxic or affect physiological activities of the cell [15]. For these reasons no probe for long-term use has been available.

Diphenyl-1-pyrenylphosphine (DPPP) is a synthetic compound with high reactivity against hydroperoxides, and has been used as a sensitive fluorescent probe for hydroperoxide analysis using HPLC postcolumn detection method [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. DPPP reduces H₂O₂ and biologically important hydroperoxides such as fatty acid hydroperoxides [17], phosphatidylcholine hydroperoxide [26], and triacylglycerol hydroperoxides [19] to their corresponding alcohols stoichiometrically. DPPP itself is not fluorescent, but DPPP oxide, resulting product of the reaction with hydroperoxides, is fluorescent with high fluorescence yield. These chemical properties, together with its hydrophobicity, seem to be advantageous when the probe is applied to the biological materials such as cell membranes. We previously reported that exogenously added hydroperoxides induced increase in fluorescence derived from DPPP in the liposomal membranes and intact cells [27]. In this study we present a detailed study to establish a new method for estimation of lipid peroxidation in the live cells using a fluorescent probe DPPP.