5-hydroxyprimaquine and Anemia--Hemolytic

Primaquine-induced hemolytic anemia is a toxic side effect that is due to premature splenic sequestration of intact erythrocytes. Previous studies have suggested that a phenolic metabolite, 5-hydroxyprimaquine (5-HPQ), mediates primaquine hemotoxicity 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 cytoskeleton, as evidenced by changes in the normal electrophoretic 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.

Topics: Anemia, Hemolytic; Animals; Antimalarials; Calcium; Cell Membrane; Cytoskeletal Proteins; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; In Vitro Techniques; Ionophores; Lipid Bilayers; Lipid Peroxidation; Male; Phosphatidylserines; Primaquine; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Spleen

Primaquine is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. Methemoglobinemia and hemolytic anemia, however, are dose-limiting side effects of primaquine therapy. These hemotoxic effects are believed to be mediated by metabolites, although the identity of the toxic specie(s) and the mechanism underlying hemotoxicity have remained unclear. Previous studies showed that an N-hydroxylated metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline, was capable of mediating primaquine-induced hemotoxicity. The present studies were undertaken to investigate the hemolytic potential of 5-hydroxyprimaquine (5-HPQ), a phenolic metabolite that has been detected in experimental animals. 5-HPQ was synthesized, isolated by flash chromatography, and characterized by NMR spectroscopy and mass spectrometry. In vitro exposure of (51)Cr-labeled erythrocytes to 5-HPQ induced a concentration-dependent decrease in erythrocyte survival (TC(50) of ca. 40 microM) when the exposed cells were returned to the circulation of isologous rats. 5-HPQ also induced methemoglobin formation and depletion of glutathione (GSH) when incubated with suspensions of rat erythrocytes. Furthermore, when red cell GSH was depleted (>95%) by titration with diethyl maleate to mimic GSH instability in human glucose-6-phosphate dehydrogenase deficiency, a 5-fold enhancement of hemolytic activity was observed. These data indicate that 5-HPQ also has the requisite properties to contribute to the hemotoxicity of primaquine. The relative contribution of N-hydroxy versus phenolic metabolites to the overall hemotoxicity of primaquine remains to be assessed.

Topics: Anemia, Hemolytic; Animals; Drug Stability; Electrochemistry; Erythrocytes; Glutathione; Hemolysis; Male; Methemoglobin; Primaquine; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds