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Iron excretion in iron excess
is also believed to contribute minimally to elimination of iron from the body, despite the fact that hepatobiliary excretion is a prominent means of excretion of other met- als such as manganese and copper.
In this study, we investigated iron excretion in Trfhpx/hpx mice, a model of inherited deficiency of the serum iron- binding protein transferrin.11 These mice develop anemia because transferrin is essential for iron delivery to ery- throid precursors. They also develop iron excess because transferrin is essential for hepcidin expression. We and oth- ers previously observed that treatment of adult Trfhpx/hpx mice with transferrin for 2 to 3 weeks not only corrected anemia and hepcidin deficiency but also lowered liver iron concen- trations.12,13 Here we exploit the latter observation to assess the effect of iron excess on iron excretion and to identify routes of iron excretion using short- and long-term trans- ferrin treatment and radioisotopic studies. Our data sug- gest that the view that iron levels are dictated solely by absorption needs to be reconsidered. They also suggest that non-gastrointestinal routes of excretion, such as exfo- liation of skin, play a minimal role in iron homeostasis.
Methods
Animals and transferrin treatment
Studies were approved by the Animal Care and Use Committee at Brown University. Mice were maintained on LabDiet 5010 containing 270 ppm iron. BALB/cJ Trf+/+ and Trfhpx/hpx mice were generated by crossing Trf+/hpx mice, which were inter- mittently backcrossed to BALB/cJ mice (Jackson Laboratories). To ensure survival of Trfhpx/hpx mice after weaning, pups were injected intraperitoneally with 3 mg human transferrin (Roche/Sigma) 2 days after birth, then once a week until wean- ing at 3 weeks of age. For all experiments, mice were aged from weaning to 2 months without transferrin injections, then some were injected intraperitoneally with 3 mg human transferrin three times a week as required for specific experiments.
Non-radioactive sample harvesting and analysis
Details on the collection of blood and tissues from mice, transferrin immunoblots, measurement of hemoglobin, hep- cidin, and RNA levels, tissue staining, and metal analysis are provided in the Online Supplementary Methods. Body and tissue iron levels were measured by inductively coupled plasma absorption emission spectrometry (ICP-AES) of acid-digested tissues in the Environmental Chemistry Facility at Brown University.
59Fe treatments, sample harvesting and analysis
To assess absorption, mice were fasted in metabolic cages (Tecniplast) with access to water for 4 h, then gavaged with 10 mCi 59FeCl3 (Perkin Elmer) and 6 mg FeCl3 in 100 mL 1 M ascorbic acid.14 The mice were then housed in metabolic cages with food and water for 16 h. 59Fe levels were measured in bodies, feces, and urine using a Triathler Gamma Counter and external NaI well-type crystal detector (Hidex). To measure body 59Fe levels, mice were anesthetized with isoflurane, placed nose-first into a 50 mL conical tube positioned vertically in the detector, and radioactivity was counted. Background counts were subtracted from all counts. Percent 59Fe absorption was calculated by expressing the sum of body and urine 59Fe levels as a percent of the sum of body, fecal, and urinary 59Fe levels.
To assess excretion, mice from the absorption studies were housed individually in regular cages for 2 months. Transferrin
treatment was continued as before when indicated. Bedding was changed once a week. Some mice were housed with a ‘buddy’ mouse not administered 59Fe. Every 1 to 2 weeks, body 59Fe levels of all the mice were measured. Buddy mouse 59Fe levels never exceeded background, suggesting that coprophagy was not prominent. Details on the conversion of body 59Fe counts to 59Fe half-lives and excretion rates are given in the Online Supplementary Methods.
To identify routes of 59Fe excretion, mice were housed overnight for 16 h in metabolic cages at least three times during the excretion study. Feces and urine were analyzed for 59Fe levels by gamma counting then for iron and ferritin levels using ICP- AES and enzyme-linked immunosorbent assay (ELISA) as described in the Online Supplementary Methods.
Mathematical modeling
The mathematical modeling of iron levels is described in the Online Supplementary Methods. A manuscript on the model is cur- rently under review and a preprint version of the paper is avail- able.15
Statistical analysis
Statistical significance (P<0.05) was calculated by a two-tailed t-test or one- or two-way analysis of variance (ANOVA) with a Holm-Sidak post-hoc test using Sigmaplot. Pearson correlations were also measured using Sigmaplot.
Results
Short-term transferrin treatment reduces tissue iron excess in Trfhpx/hpx mice
Short-term transferrin treatment of adult Trfhpx/hpx mice decreases liver iron concentrations.12,13 To investigate this phenomenon further, we first determined whether a 2- week course of transferrin treatment in 2-month old Trfhpx/hpx mice altered iron concentrations in organs other than the liver. As expected, transferrin treatment increased serum transferrin, blood hemoglobin, and serum hepcidin levels in mutant mice (Figure 1A-C). Treatment also nor- malized Fam132b RNA levels in the spleen, a site of extramedullary hematopoiesis in Trfhpx/hpx mice, with Fam132b encoding erythroferrone, which is an inhibitor of hepcidin that is expression expressed by erythroid precur- sors (Figure 1D).16 Transferrin treatment also corrects severe splenomegaly in mutant mice.12 Untreated Trfhpx/hpx mice accumulated iron largely in the liver and pancreas, specifically in hepatic periportal regions and exocrine pan- creas, and to a lesser extent in the kidneys, heart, and other tissues (Figure 1E).11 While Trf+/+ mice had stainable iron in the red pulp of the spleen, untreated Trfhpx/hpx mice had splenic iron deficiency and a paucity of stainable iron. We attribute this to the fact that hepcidin also inhibits macrophage iron export - hepcidin deficiency in mutant mice leads to persistent iron export from red pulp macrophages scavenging iron-poor red blood cells. Transferrin treatment decreased iron concentrations and tissue iron staining in the liver, pancreas, and kidneys but not in the heart or duodenum and increased iron concen- trations and tissue iron staining in the spleen (Figure 1E and Figure 2). Stainable iron was also detectable in duode- nal smooth muscle in untreated and treated Trfhpx/hpx mice but in duodenal enterocytes only in treated mutant mice (Figure 2B,C). Overall, transferrin treatment decreased iron concentrations in multiple organs in Trfhpx/hpx mice.
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