A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis.

Abstract

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

• Authors: Patrick Gutschow, Paul J Schmidt, Huiling Han, Vaughn Ostland, Thomas B Bartnikas, Michael A Pettiglio, Carolina Herrera, James S Butler, Elizabeta Nemeth, Tomas Ganz, Mark D Fleming, Mark Westerman
• Journal: Haematologica (Haematologica) Vol. 100 Issue 2 Pg. 167-77 (Feb 2015) ISSN: 1592-8721 [Electronic] Italy
• PMID: 25425686 (Publication Type: Journal Article)
• Copyright: Copyright© Ferrata Storti Foundation.

Chemical References

• Hamp protein, mouse
• Hepcidins
• Lipopolysaccharides
• RNA, Messenger
• Iron

Topics

• Anemia, Iron-Deficiency (genetics, metabolism, pathology)
• Animals
• Cells, Cultured
• Disease Models, Animal
• Enzyme-Linked Immunosorbent Assay (methods)
• Female
• Hemochromatosis (genetics, metabolism, pathology)
• Hepcidins (genetics, metabolism)
• Homeostasis (drug effects, physiology)
• Humans
• Inflammation (chemically induced, genetics, metabolism, pathology)
• Iron (administration & dosage, metabolism)
• Iron Overload (genetics, metabolism, pathology)
• Lipopolysaccharides (toxicity)
• Liver (metabolism)
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• RNA, Messenger (genetics)
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• beta-Thalassemia (genetics, metabolism, pathology)