Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

Riemer, Jan; Hoepken, Hans Hermann; Czerwinska, Hania Barbara; Robinson, Stephen R.; Dringen, Ralf

doi:10.1016/j.ab.2004.03.049

Cited by 502 publications

(442 citation statements)

References 22 publications

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“…30 In serum samples from treated and control rats, iron levels were detected by colorimetric ferrozine-based assay. 31 ELISA For detection of pro-hepcidin in serum from GD-and zymosan-treated rats, a pro-Hepcidin ELISA kit was used. 32,33 Samples were processed according to the manufacturer's instructions.…”

Section: Detection Of Serum Transaminases and Iron Levelsmentioning

confidence: 99%

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

Moriconi

Ahmad

Ramadori

et al. 2009

Laboratory Investigation

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The liver and the spleen are the organs in which cellular material and aged erythrocytes are eliminated from the blood. Within the liver, Kupffer cells (KCs) are mainly responsible for this task, as such KCs have a pivotal role in iron metabolism. The aim of this study is to investigate the changes of hepatic gene expression in two models of KC phagocytosis. Gadolinium chloride (GD) or zymosan was injected intraperitoneally into rats and to endotoxin-resistant mice (C3H/HeJ). The animals were killed at different time points and their livers were immediately frozen in liquid nitrogen for RNA isolation and immunohistological studies. RNA was analyzed by real-time PCR and northern blot. Sera were used to measure transaminases, hepcidin and iron levels. The expression of iron metabolism genes, hepcidin, hemojuvelin (Hjv), ferroportin-1 (Fpn-1) and of the inflammatory cytokines IL-6, IL-1b, TNF-a and IFN-g was determined. Although phagocytosed material was detected in ED-1-and C1q-positive cells, no inflammatory cells were identified within the liver parenchyma. Serum levels of hepcidin, iron and transaminases did not differ from those of control animals. Both GD and zymosan induced an upregulation of hepcidin-gene expression in rat liver as early as 3 h, reaching a maximum 6 h after treatment. Hjv-and Fpn-1-gene expression was downregulated at the same time. IL-6 was by far the most induced acute-phase-cytokine in GD-and zymosan-treated livers, although IL-1b and TNF-a were also strongly upregulated by zymosan and to a lesser extent by GD. Similar results were obtained in the C3H/HeJ mouse strain excluding the possible role of contaminating endotoxin. This study shows that phagocytosis upregulates hepcidin-gene expression and downregulates Hjv-and Fpn-1-gene expression within the liver. These changes in iron-regulating-gene expression may be mediated by the locally produced acute-phase-cytokines.

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Section: Detection Of Serum Transaminases and Iron Levelsmentioning

confidence: 99%

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

Moriconi

Ahmad

Ramadori

et al. 2009

Laboratory Investigation

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“…30 To measure the iron concentration, tissue homogenates were prepared as reported previously. 25 Iron was measured in both nuclear and cytoplasmic lysates from liver.…”

Section: Measurement Of Iron Levelsmentioning

confidence: 99%

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

Naz

Malik

Sheikh

et al. 2012

Laboratory Investigation

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Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1b and TNF-a) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of a2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a 'sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR. The liver is the central organ for iron metabolism. 1 Liver cells (hepatocytes) take up iron absorbed from the diet, and Kupffer cells take up iron from aged erythrocytes and distribute it to different organs. Iron is an important co-factor for oxygen transport, heme and non-heme iron proteins, electron transfer, neurotransmitter synthesis, myelin production, energy metabolism and mitochondrial function, as well as for the production of reactive-oxygen species. 2,3 As a result, its metabolism is tightly regulated. Iron homeostasis is controlled by a large group of iron-regulatory proteins. The absorption of dietary iron begins with its transport across the duodenal brush-border membrane mediated by the duodenal metal transporter-1 (DMT-1), the major iron transporte...

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“…34 Briefly, the iron bound to Tf was released in an acidic medium as ferric iron and was then reduced to ferrous iron in the presence of ascorbic acid. Ferrous iron forms a blue complex with ferrozine.…”

Section: Detection Of Serum Iron Levelsmentioning

confidence: 99%

“…Tissue homogenates were used to measure the iron levels by colorimetric ferrozine-based assay. 34 Prussian Blue Iron Staining Slides were stained with Accustain s Iron stain from SigmaAldrich using the protocol provided by the manufacturer. Briefly, the slides were deparaffinized and tissues were hydrated to deionized water.…”

Section: Preparation Of Liver Homogenate and Measurement Of Iron Concmentioning

confidence: 99%

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat

Sheikh

Dudás

Ramadori

2007

Laboratory Investigation

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Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

Cited by 502 publications

References 22 publications

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat

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