Primaquine-lnduced Hemolytic Anemia:Role of Membrane Lipid Peroxidation and Cytoskeletal Protein Alterations in the Hemotoxicity of 5-Hydroxyprimaquine

摘要

Primaquine-induced hemolytic anemia is a toxic side effect that is due to premature splenic sequestration of intact erythro-cytes.Previous studies have suggested that a phenolic metabolite,5-hydroxyprimaquine (5-HPQ),mediates primaquine he-motoxicity by generating reactive oxygen species (ROS) within erythrocytes that overwhelm antioxidant defenses.However,the nature of the oxidative stress is not understood,and the molecular targets,whether protein and/or lipid,are unknown.To investigate the mechanism underlying the hemolytic activity of 5-HPQ,we have examined the effect of hemolytic concentrations of 5-HPQ on ROS formation within rat erythrocytes using the cellular ROS probe,2',7'-dichlorodihydrofluoresein diacetate.In addition,we examined the effect of 5-HPQ on membrane lipids and cytoskeletal proteins.The data indicate that 5-HPQ causes a prolonged,concentration-dependent generation of ROS within erythrocytes.Interestingly,5-HPQ-generated ROS was not associated with the onset of lipid peroxidation or an alteration in phosphatidylserine asymmetry.Instead,5-HPQ induced oxidative injury to the erythrocyte cy-toskeleton,as evidenced by changes in the normal electro-phoretic pattern of membrane ghost proteins.Immunoblotting with an anti-hemoglobin antibody revealed that these changes were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts.The data suggest that cytoskeletal protein damage,rather than membrane lipid peroxidation or loss of phosphatidylserine asymmetry,underlies the process of removal of erythrocytes exposed to 5-HPQ.