DETECTION OF LIPID-PEROXIDATION ON ERYTHROCYTES USING THE EXCIMER-FORMING PROPERTY OF A LIPOPHILIC BODIPY FLUORESCENT DYE

Lipophilic analogues of dipyrrometheneboron (BODIPY-FL) dyes, used for membrane studies, normally fluoresce in the green wavelength region ( similar to 516 nm), but at high local concentration; they shift their emission to the red region (similar to 540-600 nm) via excimer formati on. A two-wavelength-based method is described that utilizes the excim er-forming property of BODIPY-FL for the sensitive monitoring of membr ane lipid peroxidation on erythrocytes (RBCs). Bodipy-FL-C-3-EDA, a re latively water-soluble analogue of BODIPY-FL, loses its single fluores cence peak at 516 nm upon reaction with peroxyl radicals generated in aqueous phase by 2,2'-azobis(2-amidinopropane), AAPH, and the loss of fluorescence is prevented by the presence of the peroxyl radical scave nger, Trolox (a water-soluble analogue of vitamin E). Hexadecanoyl-BOD IPY-FL (C-16-BODIPY, a lipophilic analogue of BODIPY-FL) incorporated into RBC membranes, in addition to the peak at 516 nm, also forms exci mer fluorescent peaks at 546 and 590 nm; The relative intensity of the emission peaks depends on the concentration of membrane-incorporated dye. Upon addition of cumene hydroperoxide (CH, 0-10 mu M) or benzoyl peroxide (BP, 0-5 mu M) to C-16-BODIPY-labeled RBC suspensions, gradua l changes in the fluorescent peaks occur; the 516 nm peak initially in creases, then decreases, while the 546 and 590 nm excimer peaks contin uously decrease, as measured by fluorometry or by flow cytometry. The data indicate that lipid peroxidation radicals reacting with C-16-BODI PY localized on RBC membranes oxidize the dye and the resulting molecu le cannot participate in the excimer formation; this oxidization leads to the observed changes in the fluorescent peaks. The ratio of the fl uorescence levels at 590 and 516, nm is a measure of the excimer forma tion between fluorophores and can be used to monitor the onset of lipi d peroxidation in RBCs. (C) 1997 Elsevier Science B.V.