Abstract/Summary

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05–0.2 μM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N ׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis. 1. Introduction The oxidation of low density lipoprotein (LDL) has been proposed to occur in the extracellular space of the arterial wall and lead to the formation of foam cells and atherosclerosis (Steinberg, 2009). The oxidation of LDL by cells requires the presence of micromolar concentrations of the transition metals copper or iron in the medium (Steinbrecher et al., 1984; Leake and Rankin, 1990). Free copper or iron are not readily available in the plasma or interstitial fluid because they exist in a tightly bound form. A number of mechanisms have been proposed to be involved in the oxidation of LDL in vivo, but at present, there is no consensus on the predominant mechanism by which LDL is modified in vivo. Cultured macrophages have been shown, however, to take up aggregated or acetylated LDL quickly and oxidise it in lysosomes (Wen and Leake, 2007). Cholesterol crystals derived from oxidised LDL in lysosomes have been reported to rupture these organelles in macrophages and activate the NLRP3 inflammasome (Duewell et al., 2010). This might be important as atherosclerosis is a chronic inflammatory disease and