An esterification protocol for cis‐parinaric acid‐determined lipid peroxidation in immune cells1,2

Loss of fluorescence from cis‐parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe2+‐EDTA resulting in a dose‐dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe2+‐induced lipid peroxidation. In separate experiments, 20 μM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37°C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 μM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100‐fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose‐dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co‐incubation with α‐tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content.