NOVEL FLUORESCEIN-BASED FLOW-CYTOMETRIC METHOD FOR DETECTION OF LIPID-PEROXIDATION

The novel property of fluorescein to detect peroxyl radicals is demons trated. On the basis of this observation, a fluorescein-based, flow-cy tometric method to directly and continuously detect free radicals gene rated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein -labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-az obis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolo x. When 5-/6-CF free in solution is incubated with red blood cells exp osed to cumene hydroperoxide (CH), a similar loss of fluorescence occu rs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluo rescein (C-11-fluor), a lipophilic fluorescein conjugate, has been inc orporated into the membranes of RBC. Upon addition of CH, a decrease i n fluorescence is fluorometrically observed that is proportional to th e amount of hydroperoxide added and inhibited by preincubation with Tr olox or vitamin E. Flow-cytometric studies are then performed to demon strate that C-11-fluor can monitor free radicals generated during lipi d peroxidation on a cell-by-cell basis. When exposed to CH, a time-dep endent shift of the flow-cytometric profile toward lower values is obs erved that is inhibited by Trolox or vitamin E. This approach in conju nction with multiparametric flow cytometry may allow examination of th e biologic significance of lipid peroxidation by correlation to other cellular end points on single cells. Copyright (C) 1996 Elsevier Scien ce Inc.