Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload
Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload
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DOI：
10.1136/gut.50.3.413
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发表时间：
2002-03-01
期刊：
影响因子：
24.5
通讯作者：
Niitsu, Y
中科院分区：
文献类型：
--
作者：
Miyazaki, E;Kato, J;Niitsu, Y
背景和目的：铁以铁蛋白和含铁血黄素的形式储存在肝细胞中。在铁超载的肝脏中，富含铁黄素的含量显著增加，超过铁蛋白和铁黄素结合能力的铁离子可能会促进自由基的产生，导致细胞损伤。本研究的目的是用四种针对天然和变性H/L铁蛋白亚基的抗体来鉴定肝含铁血黄素。方法：从3例输血后铁负荷过高的患者的肝脏中制备铁蛋白和铁黄素。用非变性聚丙烯酰胺凝胶电泳法(PAGE)-免疫印迹法对组装的铁蛋白分子进行分析。用变性十二烷基硫酸钠(十二烷基硫酸钠)-PAGE-免疫印迹分析铁蛋白亚基。结果：非变性PAGE-免疫印迹分析表明，天然铁蛋白抗体能识别组装的肝脏铁蛋白，变性亚基抗体不能识别组装的肝脏铁蛋白。SDS-PAGE免疫印迹和免疫金电镜分析均显示，铁负荷肝脏的铁血黄素主要与变性H-铁蛋白亚基的单抗反应，与变性L铁蛋白的反应程度较小，与天然H-铁蛋白或L铁蛋白的抗体反应很弱。结论：铁负荷肝脏中的铁血黄素主要由变性H-铁蛋白亚基组成。
Background and aims: Iron is stored in hepatocytes in the form of ferritin and haemosiderin. There is a marked increase in iron rich haemosiderin in iron overloaded livers, and ferric iron in amounts exceeding the ferritin and haemosiderin binding capacity may promote free radical generation, causing cellular damage. The aim of this study was to characterise hepatic haemosiderin using four antibodies specific for either native or denatured H/L-ferritin subunits.Methods: Ferritin and haemosiderin were prepared from the livers of three patients with post-transfusional iron overload. The assembled ferritin molecules were analysed by non-denaturing polyacrylamide gel electrophoresis (PAGE)-immunoblotting. Ferritin subunits in the haemosiderin fraction were assessed by denaturing sodium dodecyl sulphate (SDS)-PAGE-immunoblotting. Distribution of native and denatured ferritin subunits in hepatocytes was examined by immunogold electron microscopy.Results: Non-denaturing PAGE-immunoblot analyses showed that the assembled liver ferritins were recognised by the antibodies for native ferritins and not by those for the denatured subunits. Both SDS-PAGE-immunoblot and immunogold electron microscopic analyses disclosed that haemosiderin of iron overloaded liver reacted predominantly to the monoclonal antibody for the denatured H-ferritin subunit, to a lesser degree to that for denatured L-ferritin, and very weakly, if any, with antibodies for native H-ferritin or L-ferritin.Conclusions: These results suggest that in iron overloaded liver, haemosiderin consists predominantly of denatured H-ferritin subunits.