This proposal describes a plan to study the role of infection mediated oxidant damage in sickle cell anemia (SCA). Infections are known to precipitate hyperhemolysis and vaso-occlusive crisis in SCA. I plan to determine if oxidants released from neutrophils during infection alter the red cell membrane and thereby contribute to these clinical events. The first phase of my study will utilize sensitive techniques to detect in vivo evidence for red blood cell (RBC) oxidant damage during infection. These techniques include high performance liquid chromatography to detec lipid hydroperoxides, fluorescent spectroscopy to detect malondialdehyde cross-linked lipids or proteins and diene conjugation to detect peroxidized lipids. Relationships between cell density and oxidant damage will be explored. I will next perform in vitro studies which utilize activated neutrophils (AN) incubated with RBCs to determine if structural changes are induced in sickle cell membranes by oxy radicals elaborated from AN. In addition to standard techniques to measure oxidant damage of lipids and proteins, I will use electron spin resonance and spin labeling techniques to study lipid/protein alterations and thiol-disulfine exchange chromatography to measure sulfhydryl group oxidation. These studies will also incorporate use of different radical scavengers to identify products of AN involved in oxidation. Functional changes (effects of AN on membrane enzyme activities, cation flux, cell density and deformability) which may accompany structural damage induced by AN will be studied. To correlate oxidant damage with possible alterations in cell surface properties, the effects of AN on adherence of RBC's to monocytes will be measured. The last phase of this study will investigate the mechanisms of AN induced oxidant damage. I will employ two model systems: hemosomes [hemoglobin (Hb) S or A encapsulated into liposomes] and hybrid erythrocytes (normal membranes/sickle Hb; sickle membranes/normal Hb). These systems will permit manipulations of Hb, lipid content, free radical scavengers and membrane components in order to elucidate the role of each in susceptibility of sickle RBC to AN mediated damage. In summary, this project will 1) look for in vivo evidence of RBC oxidant damage in infected SS patients, 2) define membrane structural damage caused by AN and correlate structural damage with resultant functional cellular changes which may predispose to exacerbations of SCA, and 3) investigate the mechanisms of AN induced oxidant damage.
