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Title: The role of ceruloplasmin in brain iron transport
Authors: Tsoi, Ying-king
Degree: M.Phil.
Issue Date: 2000
Abstract: This research project aims to study the role of Ceruloplasmin (CP) in brain iron transport by the use of transferrin-free iron (59Fe2+) and glioma cell line BT325. CP is a copper-binding protein suggested functioning in iron metabolism. In this study, rat CP was isolated successfully by the modified protocol (Ryan et al. 1992). The isolated rat CP has an A610/A280 ration of 0.044 (indicates the purity is near 100%) and an oxidase activity of 239.8 units/ml (using N,N-dimethyl-p-phenylenediamine as substrate) (Chapter 3). The isolated rat CP was used for the investigation of the species-specificity of CP functions in brain iron transport. The iron stimulatory effect of CP was not species-specific, at least among the three CPs (human CP, rat CP and bovine CP) tested (Chapter 6). The Fe2+ uptake properties of the BT325 cells were also characterized (Chapter 4). The optimum pH was in the range of 5.5-6.0. The uptake increased with a rise in iron concentration (0-5 uM) and showed saturation, giving a Vmax of 7.64 pmole Fe/mg protein and mean Km of 0.05uM. Also, three buffers (0.32 M sucrose, 0.155 M sodium chloride solutions and the maintenance medium of the cell line, RPMI 1640 medium) were used for buffer selection. At low iron concentration (1 uM Fe2+), no significant difference in the iron uptake was observed. A 30-minutes incubation time was chosen for subsequent experiment, as iron uptake was stable in this condition. In Chapter 5, the effect of CP on the iron flux of brain cells was investigated. CP did not aid in iron release in iron-loaded glioma cells. Although iron release was observed when apoTf (20 and 50 ug/ml) was present in the incubation mixture, the effect was not due to CP but apoTf. Therefore, it is concluded that CP did not involve in the iron release of this glioma cells. To further investigate the role of CP in iron flux, the effect of CP on iron uptake was studied (Chapter 7). By incubating the cells with (0-300 ug/ml) CP and 1 uM Fe2+ at 37°C for 30 minutes, iron uptake was increased significantly. The minimal effective CP concentration had to be more than 5ug/ml and the maximal uptake was reached at 30 ug/ml. The results demonstrated that CP helped in iron uptake but not release, and it plays a role in brain iron metabolism. Since there are reports in which CP stimulated iron uptake was only observed in iron-deficient cells, the effect of CP on iron uptake of iron-deficient BT325 was studied (Chapter 8). Two groups of iron-deficient glioma cells were prepared by incubating them with 2 mM Fe3+ chelator desferoxamine mesylse (DFO) and 0.5 mM Fe2+ chelator bathophenanthroline disufonic acid (BP) for 16 hours in serum-free RPMI medium at 37°C. Before this prepatation, the effect of these iron chelators on the growth of this cell line was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium bromide (MTT) assay. It was showed that an incubation time longer than one day with these two iron chelators inhibited the growth of these cells. The incubation of 0-30 ug/ml CP and 1 uM Fe2+ with both the iron-sufficient and iron-deficient cells for 30 minutes at 37 ℃ demonstrated that there was no significant difference between the iron uptake of these two groups. These results are not consistent with recent findings that CP is able to stimulate iron uptake only in iron-deficient cells. This may be due to differences in experimental conditions and the use of cell type. In addition, the importance of CP ferroxidase activity on iron uptake of BT325 cells was supported by the use of ferroxidase-defective CPs, apoCP and heat-inactivated CP which did not stimulate iron uptake. Finally, CP-stimulated iron uptake was shown to be inhibited by both 10 uM divalent and trivalent cations (Fe2+, Cu2+, Mg2+, Zn2+, Ca2+, Al3+ and Cr3+). This raises the suggestion that these metals may be transported by the same mechanism which needs further investigation.
Subjects: Brain -- Physiology
Iron -- Metabolism
Hong Kong Polytechnic University -- Dissertations
Pages: 198 leaves : ill. ; 30 cm
Appears in Collections:Thesis

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