The ferroxidase Hephaestin is required for optimal iron absorption in weanling mice, but is dispensable for enhanced iron absorption in adults
Authors: Doguer Caglar , Ha Jung‐Heun , Alla Martin , Collins James Forrest 
Published: November 2023

IntroductionHumans have no regulated means to rid the body of excess iron. Overall body iron levels are thus determined by intestinal iron absorption. Absorption of non‐heme, ferric iron by duodenal enterocytes requires enzymatic reduction and subsequent import by DMT1. Ferrous iron is then exported by the basolateral iron transporter FPN1. After export, ferrous iron can be oxidized by hephaestin (HEPH) to allow ferric iron to bind to transferrin (TF) in the interstitial fluids. HEPH is the only identified intestinal ferroxidase (FOX), but recent studies have questioned its essentiality. Moreover, we recently demonstrated that other intestinal ferroxidases exist. The current study was thus designed to test the hypothesis that intestinal HEPH is required for iron absorption only under conditions in which intestinal iron absorption is enhanced.MethodsWe initiated 3 studies to assess intestinal iron absorption in male and female, intestine‐specific HEPH KO mice (HEPHint/y) and WT littermates. As follows: Study 1‐ dietary iron deprivation; Study 2‐ acute hemolytic stress; Study 3‐ during the rapid post‐natal growth period. For study 1, adult mice of both genotypes were fed a control (50 ppm) or low‐iron (3–5 ppm) Fe diet for 6 weeks. In study 2, adult mice were injected IP with a hemolytic agent (phenylhdrazine [PHZ]) to stimulate erythropoiesis. In study 3, iron absorption was assessed in weanling mice of both genotypes and sexes. For all studies, intestinal iron absorption and whole‐body iron utilization were tested by oral gavage of 59Fe followed by calculation of iron absorption levels and tissue iron distribution, using a standard protocol. All results reported below achieved statistical significance (except where noted; One‐Way ANOVA) and numbers of animals studies varied between 5 and 12 per group in each experiment.ResultsAdult HEPHint/y KO mice were iron deficient but not anemic. Hemoglobin (Hb) levels were, however, depressed in adult mice fed the low‐iron diet or injected with PHZ regardless of genotype or sex. Contrary to published reports, we noted no defect in iron absorption in adult mice lacking intestinal HEPH under basal conditions or after dietary iron deprivation or acute hemolytic stress. Both genotypes and sexes were able to appropriately upregulate iron absorption in response to these stressors. Conversely, male and female weanling HEPHint/y KO mice were iron‐deficient and anemic, consistent with earlier reports. At this age, iron absorption was depressed in female KO mice while it was similar in males of both genotypes. Since increased iron absorption was anticipated due to the anemia, an impairment of iron absorption was apparent in both sexes at weaning.ConclusionsIntestinal HEPH is required for optimal iron absorption during the rapid growth phase of early life but is dispensable in adults subjected to low‐iron or hemolytic stress. Given that all these conditions require enhanced iron absorption, it is not immediately clear why HEPH is required only under some of these conditions. We postulate that other intestinal or circulating (e.g. ceruloplasmin) FOXs may compensate for lack of intestinal HEPH in adult mice.Support or Funding InformationNIH grant 1R21 DK068349