Mechanism of in vitro heme-induced LDL oxidation: effects of antioxidants

Background Heme protein toxicity, owing to generation of reactive oxygen species most likely by direct interaction between heme iron and hydrogen peroxide (H 2 O 2 ), may be involved in various pathologies, including atherogenesis and pigmentary acute renal failure. The aim of this study was to investigate the mechanism of heme cytotoxicity and the effects of antioxidant therapies in an in vitro model of heme-induced low-density lipoprotein (LDL) oxidation. Materials and methods Human LDLs were exposed to heme, iron (Fe), protoporphyrin (PPIX) and PPIX-Zinc (Zn) with or without H 2 O 2 . Lipid peroxidation was monitored by measurement of conjugated diene formation (at the 234-nm absorbance). The effect of various antioxidants, such as vitamin E and vitamin C, reduced glutathione (GSH), and oxidized glutathione (GSSG), mannitol and desferoxamine (DFO) was further investigated in the established in vitro model of LDL oxidation. Results Incubation of LDLs in the presence of heme/H 2 O 2 induced lipid peroxidation with the optimal oxidation rate being at 5 μM heme/100 μM H 2 O 2 doses. By contrast, incubation of LDL with H 2 O 2 , Fe, Fe/H 2 O 2 , PPIX, PPIX/H 2 O 2 , heme or PPIX-Zn did not initiate any LDL oxidation. In vitro, the vitamin E load protected native LDLs against heme/H 2 O 2 oxidative modifications. Incubation of LDLs with increasing doses of vitamin C, GSH and DFO conferred a dose-dependent protection, while mannitol and GSSG had no effect. Conclusions Initiation and propagation of heme-induced lipid peroxidation is not mediated by a Fenton reaction but depends on specific interactions between heme and H 2 O 2 . It may result from the generation of ferryl and perferryl radicals derived from hemic Fe and H 2 O 2 interactions. A protective effect of vitamins E, C, GSH and DFO was demonstrated in this model.