Background Many lines of evidence support the role of oxidised low density lipoprotein (LDL) as the main culprit in atherogenesis. Our laboratory has previously shown that iron is involved in the oxidation of LDL in lysosomes, a possible mechanism for the progression of atherosclerosis. Ferritin is an iron-storage protein which might enter lysosomes by autophagy and be involved in lysosomal LDL oxidation. The aim of this study was to test the hypothesis that ferritin can catalyse the oxidation of LDL at lysosomal pH and that antioxidants protect against this oxidation.
Method LDL (50 ug protein/ml) was oxidised by ferritin (0.05–0.2 uM) at 37oC and pH 4.5, the formation of conjugated dienes was monitored spectrophotometrically at 234 nm up to 1200 min. The effect of pH on LDL oxidation was tested by comparing the oxidation of LDL by ferritin (0.1 and 0.2 uM) at pH 4.5 or pH 7.4. The iron released from ferritin was monitored spectrophotometrically using the ferrous iron chelator bathophenanthroline. Ferritin (0.1 uM) was incubated at 37oC at pH 4.5 and pH 7.4. Bathophenanthroline (30 uM) was added at different time intervals up to 24 h and the amount of ferrous complex was measured at 535 nm. Iron release was also measured by ultrafiltration followed by atomic absorption spectrophotometry.
Results LDL was oxidised effectively by ferritin (0.05–0.2 uM). The oxidation was much faster at lysosomal pH 4.5 than at pH 7.4, which could be attributed to our finding that more iron was released from ferritin at pH 4.5. EDTA and diethylenetriamine pentaacetate inhibited the oxidation, but did not inhibit it entirely. The water-soluble lysosomotropic drug cysteamine (5 uM-10 mM) inhibited the initial oxidation of LDL in a concentration-dependent manner, although the lower concentrations exhibited a delayed prooxidant effect which was less marked with the higher concentrations. Concentrations above 1 mM had no prooxidant effect. Cysteamine was shown, using the ferrous iron chelator bathophenanthroline, to release iron from ferritin and this might explain the prooxidant effect. The lipid-soluble antioxidant N, N′-diphenyl-p-phenylenediamine (5 and 10 uM) inhibited the oxidation of LDL by ferritin without any prooxidant effect.
Conclusion These findings support the possible involvement of ferritin in lysosomal LDL oxidation and the use of appropriate antioxidants to prevent this oxidation in atherosclerosis.
Acknowledgement We would like to thank the Tertiary Education Trust Fund (TETFund) of the Federal Republic of Nigeria for funding this project.
- Low density lipoprotein
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