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The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

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 (Hbbth3/+), hereditary hemochromatosis (Hfe−/−, Hjv−/−, and Tfr2Y245X/Y245X), hypotransferrinemia (Trfhpx/hpx), heterozygous transferrin receptor 1 deficiency (Tfrc+/−) and iron refractory iron deficiency anemia (Tmprss6−/− and Tmprss6hem8/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.

Mutagenesis screens can establish mouse models of utility for the study of critical biological processes such as iron metabolism. Such screens can produce mutations in novel genes or establish novel alleles of known genes, both of which can be useful tools for study. In order to identify genes of relevance to hematologic as well as other phenotypes, we performed N-ethyl-N-nitrosourea mutagenesis in C57BL/6J mice. An anemic mouse was identified and a putative mutation was characterized by mapping, sequencing and in vitro activity analysis. The mouse strain was backcrossed for ten generations then phenotypically characterized with respect to a previously established null mouse strain. Potential modifying loci were identified by quantitative trait locus analysis. Mapping and sequencing in an anemic mouse termed hem8 identified an I286F substitution in Tmprss6, a serine protease essential for iron metabolism; this substitution impaired in vitro protease activity. After backcrossing to C57BL6/J for ten generations, the hem8−/− strain exhibited a phenotype similar in some but not all aspects to that of Tmprss6−/− mice. The hem8 and Tmprss6-null mutations were allelic. Both hem8−/− and Tmprss6−/− mice responded similarly to pharmacological modulators of bone morphogenetic protein signaling, a key regulator of iron metabolism. Quantitative trait locus analysis in the hem8 strain identified potential modifying loci on chromosomes 2, 4, 7 and 10. In conclusion, the hem8 mouse model carries a novel allele of Tmprss6. Potential uses for this strain in the study of iron metabolism are discussed.

Here we investigate the regulation of hepcidin, a hormone that inhibits dietary iron absorption and macrophage iron recycling, by the serum iron-binding protein transferrin. Mice deficient in transferrin (Tfhpx/hpx) and hemojuvelin (Hjv−/−), a gene mutated in juvenile hemochromatosis, a disease of hepcidin deficiency and iron overload, were generated. While Tfhpx/hpx Hjv+/+ and Tfhpx/hpx Hjv−/− phenotypes did not differ markedly, transferrin treatment and RBC transfusions robustly increased hepcidin levels in Tfhpx/hpx Hjv+/+ but not Tfhpx/hpx Hjv−/−mice. These results suggest that, while hemojuvelin is not essential for the establishment or maintenance of hepcidin deficiency in transferrin-deficient mice, hemojuvelin is essential for transferrin-dependent and tansferrin-independent hepcidin expression in conditions of iron overload.