Investigation of the release of iron from ferritin by naturally occurring antioxidants
Introduction
Ferritin is the principal protein involved in storing iron in a soluble, non-toxic, yet bioavailable form in bacteria, plants and animals where it maintains up to 4500 atoms of hydrolysed and polymerised iron(III) atoms in a soluble form within a protein shell [1]. Mammalian ferritins consist of a protein shell with twenty-four subunits surrounding a core of ferrihydrate, each subunit weighing either 19 766 Da (the L subunit) or 21 099 Da (the H subunit) [2], [3]. In normal human subjects 25% of the total body iron is found in the storage protein ferritin. Brain ferritin can have 60–70% H subunit content while horse spleen ferritin and mammalian liver ferritin are predominantly L subunits. Brain ferritin has been reported to have an iron saturation of 1500 iron atoms per ferritin molecule, half that of liver or spleen ferritin [4], [5].
When high levels of toxic metals enter the brain, ferritin may even play an important role in ameliorating metal toxicity. The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Ferritin iron may be released by a variety of reducing agents arising from their ability to reduce tightly bound iron(III) to the more mobile iron(II) form [6], [7]. Iron(II) released is often detected by the formation of the coloured complex with ferrozine [8], [9]. Iron mobilisation from ferritin can also proceed by direct chelation of iron(III), this may or may not be followed by reduction to iron(II). In the absence of chelators, the reduced core of ferritin iron appears to remain within the protein shell [10]. Strong chelators acting alone can remove iron from ferritin but this occurs extremely slowly [11]. As a result it would be expected that the most efficient reagents would be those whose oxidised products can complex with the iron(II) produced when they react with the bound iron(III) [9]. Sinapic acid and ferulic acid are known antioxidants and can be effective in preventing lipid damage [12]. Unlike most phenolic based compounds previously investigated for their kinetic/mechanistic reactions with iron(III), spectral data provided no evidence for the formation of an iron–ferulic acid or sinapic acid complexes, strongly suggesting that in both these cases electron transfer proceeds via an ‘outer-sphere’ reaction [13]. It was therefore expected that these phenolic compounds would be able to facilitate removal of iron from sources such as ferritin by means of prior reduction of the iron(III) to iron(II). This proposal has a precedent in the case of 6-hydroxydopamine, which is selectively toxic to catecholaminergic neurons [9], [11]. Studies aimed at modelling the pathogensis of Parkinson’s disease demonstrated that 6-hydroxydopamine is highly effective in releasing iron as iron(II) from ferritin, and also, unlike other catecholamines, reacts with iron(III) in a completely ‘outer-sphere’ fashion [9].
Numerous epidemiological studies have associated a significant negative correlation between the intake of tea and serum ferritin [14], [15]. Major constituents of green tea include polyphenolic compounds such as epigallocatechin and gallic acid methyl ester, which together with similar phenolic compounds can comprise up to 40% (w/w) of green tea. Linert and Jameson [11] concluded that ligands capable of releasing iron from ferritin must be capable of reducing ferritin bound iron(III) while the oxidised form of the ligand must also be capable of complexing iron(II), properties possessed by this family of compounds [16], [17].
Our initial studies have concentrated on the simpler phenols and we now report the results of our kinetic studies of the release of iron from horse spleen ferritin by ferulic acid, sinapic acid, gallic acid methyl ester and epigallocatechin.
Section snippets
Experimental
Horse spleen ferritin (69 mg/ml ferritin) was purchased from Sigma. The purity of the sample was assessed by SDS–PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) on a 12.5% total acrylamide gel according to the method of Laemmli [18]. Densitometric analysis on a FluorS MultiImager using multianalyst software confirmed the purity of the Sigma preparation to be ∼95%. Atomic absorption analysis of a sample gave an iron content of 4.6%. This corresponds to ∼384 atoms of iron per
Results
Fig. 1 shows the time dependant release of iron from ferritin on addition of an excess of gallic acid methyl ester as monitored spectrophotometrically by formation of the iron ferrozine complex. Similar results were obtained for the sinapic and ferulic acids and epigallocatechin. Fig. 2 shows the percentage of the bound iron released on reacting ferritin with various concentrations of epigallocatechin, gallic acid methyl ester, sinapic acid and ferulic acid for 30 min. The order of ability to
Discussion
Consumption of polyphenol rich foods such as green tea interferes with iron metabolism through decreasing absorption due to the formation of strong complexes, which can solubilise and remove iron from the body. In the present investigation it was observed that the presence of epigallocatechin, gallic acid methyl ester, sinapic acid and ferulic acid induced iron release from ferritin. This is in agreement with numerous epidemiological studies which linked the intake of polyphenol rich foods with
Acknowledgements
The work is supported by Enterprise Ireland Basic Research Grant SC/1998/487. One of us (MOC) thanks Enterprise Ireland for a Basic Research Award. We gratefully acknowledge the assistance of Dr. Maria Touhy of the Department of Biochemistry who carried out the SDS–PAGE analysis of the ferritin.
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2018, Biomedicine and PharmacotherapyCitation Excerpt :The images were analyzed using the formula: % area = stained area / (parenchymal area − blank area) × 100 [37,38]. Ferrozine reagent was used to measure the release of iron from ferritin at 560 nm using a spectrophotometer according to the previously reported protocol [39]. The western blotting analysis was performed to observe the effects of EA treatment on iron-overloaded liver samples of Swiss albino mice.