AN ESTERIFICATION PROTOCOL FOR CIS-PARINARIC ACID-DETERMINED LIPID-PEROXIDATION IN IMMUNE CELLS

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive ind icator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether en richment of membranes with polyunsaturated fatty acids (PUFA) increase d lipid peroxidation. P388D1 macrophages were labeled by addition of c PnA as an ethanolic solution. Within two minutes of addition, in the a bsence of serum, cPnA rapidly intercalated into the plasma membrane. L ipid peroxidation was initiated by addition of Fe2+-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant conce ntration. Cells previously enriched with PUFA and labeled by intercala tion showed no differences in spontaneous or Fe2+-induced lipid peroxi dation. In separate experiments, 20 mu M cPnA in ethanolic solution wa s injected into cell culture media containing 0.1% essentially fatty a cid free bovine serum albumin (BSA). Cells were resuspended and incuba ted for 90 min at 37 degrees C. After washing with BSA to remove-cPnA which had not incorporated, 0.5% (0.1 mu M) of the added cPnA was foun d esterified within cellular lipids. This level of cPnA provided a 100 -fold increase over basal autofluorescence levels. Cells labeled in th is manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by e sterification had significantly increased rates and total amounts of l ipid peroxidation. Co-incubation with alpha-tocopherol and PUFA result ed in a decrease in lipid peroxidation which was not significantly dif ferent from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxid ation induced by alteration of membrane PUFA and/or vitamin E content.