Denatured H-ferritin subunit is a major constituent of haemosiderin in the liver of patients with iron overload

• Published in: Gut Vol. 50; no. 3; pp. 413 - 419
• Main Authors: Miyazaki, E, Kato, J, Kobune, M, Okumura, K, Sasaki, K, Shintani, N, Arosio, P, Niitsu, Y
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group Ltd and British Society of Gastroenterology 01.03.2002; BMJ; BMJ Publishing Group LTD; Copyright 2002 by Gut
• Subjects: Adult; Antibodies, Monoclonal - immunology; Antibody Specificity; Apoferritins; Biological and medical sciences; bovine serum albumin; BSA; CBB; Coomassie Brilliant Blue R-250; ferritin; Ferritins - analysis; Ferritins - immunology; haemosiderin; Hemosiderin - analysis; Hepatocytes - chemistry; Hepatocytes - ultrastructure; Humans; Hypotheses; immunoelectron microscopy; Immunoglobulins; IRE; iron overload; Iron Overload - metabolism; Iron Overload - pathology; iron responsive elements; Liver - chemistry; Liver Disease; Male; Medical sciences; Metabolic diseases; Metals (hemochromatosis...); Microscopy; Middle Aged; MoAb; monoclonal antibody; Other metabolic disorders; PAGE; Patients; PBS; phosphate buffered saline; polyacrylamide gel electrophoresis; Protein Denaturation; Proteins; recombinant H-ferritin; recombinant L-ferritin; rHF; rLF; SDS; sodium dodecyl sulphate; Human; Overload; Liver; Ferritin; Digestive diseases; Hepatic disease; Metabolic diseases; Iron
• ISSN: 0017-5749; 1468-3288; 1458-3288
• DOI: 10.1136/gut.50.3.413

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.