Intestinal iron uptake determined by divalent metal transporter is enhanced in HFE- deficient mice with hemochromatosis  William J.H. Griffiths, Timothy M. Cox  Gastroenterology  Volume 120, Issue 6, Pages 1420-1429 (May 2001) DOI: 10.1053/gast.2001.24050 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Mucosal uptake of ferrous iron compared between 6 matched pairs of HFE-knockout and wild-type mice. Proximal small intestine from each animal was divided into 24 equal slices and iron uptake determined at 6 ferrous iron concentrations in quadruplicate. Uptake of ferrous iron was increased at all 6 concentrations in the HFE-deficient mice compared with paired wild-type controls (P < 0.0001; Wilcoxon stratified rank sum test). Gastroenterology 2001 120, 1420-1429DOI: (10.1053/gast.2001.24050) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of Fe2+-chelation on uptake of iron presented to mucosa as Fe3+. Iron uptake was measured at low and high concentrations of ferric iron in the presence or absence of 0.5 mmol/L ferrozine in the incubation medium. Proximal small intestine from each of 3 wild-type mice was divided into 20 equal slices, and 5 intestinal samples randomly were allocated to each assay tube. Uptake of 3.5 μmol/L ferric iron was inhibited by 75% (P < 0.05) compared with 30% inhibition at 450 μmol/L. Gastroenterology 2001 120, 1420-1429DOI: (10.1053/gast.2001.24050) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Identification and quantification of immunoreactive DMT1 protein in duodenal mucosal extracts derived from HFE-knockout and wild-type mice. (A) Polyclonal antibodies raised against murine DMT1 peptide were analyzed by Western immunoblotting. Lane 1 shows a representative mucosal extract from the wild-type group probed after electrophoresis with rabbit antiserum to DMT1; the immunoreactive signal was developed with an HRP-conjugated anti-rabbit immunoglobulin. Lane 2 shows an extract from an HFE-deficient animal with equivalent protein concentration (5.6 mg/mL) and equal loading (10 μL) probed identically. In both lanes a single immunoreactive species was identified at approximately 60 kilodaltons. In control lanes 3 and 4, wild-type mucosal extract was electrophoresed and probed respectively with preimmune serum and with rabbit anti-serum to DMT1 preincubated with DMT1 peptide. No species were identified in lane 3, and a faint signal corresponding to DMT1 polypeptide was observed in lane 4. Lane 5 shows a separate blot with a single immunoreaction at 60 kilodaltons between chicken antiserum to DMT1 and wild-type mucosal extract. In the negative control (lane 6) wild-type mucosal extract was probed with preimmune chicken serum from the same animal. (B) Standard curve of ELISA using DMT1 peptide and rabbit antiserum specific to DMT1. (C) Angular transformation of standard curve, arcsine ✓ (b/b0) where b represents the response variable (optical density) and b0 the maximum immunoreaction derived from the positive control wells. The horizontal axes in B and C were normalized for the presence of measured amounts of competing antigen, 3.1 nmol of which was added to the first well and diluted serially for calibration. Gastroenterology 2001 120, 1420-1429DOI: (10.1053/gast.2001.24050) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of specific antibody to DMT1 on ferrous iron uptake. (A) Proximal small intestine from each wild-type mouse (n = 4 animals) was divided into 10 equal slices; 5 were randomly allocated to incubation for 3 hours at room temperature with 1.2 mg/mL antibody to DMT1 (this was presented as IgG purified from the yolks of eggs laid by a single chicken after immunization with the DMT1 peptide conjugate described in Materials and Methods) in oxygenated HEPES buffer; 5 additional slices were incubated for 3 hours with 1.2 mg/mL preimmune avian IgG that was purified from eggs laid by the same animal before immunization. After immunoglobulin treatment, the intestinal slices were washed vigorously on 3 occasions for 5 minutes in HEPES buffer at room temperature. Uptake of 18 μmol/L ferrous iron was substantially inhibited after preincubation with antibody to DMT1 (P < 0 01). (B) Kinetics of ferrous iron uptake were reassessed between 3 matched pairs of HFE-knockout and wild-type mice. ♦, Wild-type; ■, HFE-KO. Twenty-four intestinal slices from each HFE-knockout mouse were preincubated for 3 hours at room temperature with avian antibody to DMT1 (1.2 mg/mL) before calculating ferrous iron uptake at each of 6 concentrations in quadruplicate. Intestinal samples from each wild-type mouse were preincubated similarly with preimmune control IgG (1.2 mg/mL) before uptake kinetics were measured. Ferrous iron uptake was significantly lower in HFE-deficient animals exposed to DMT1 antibody compared with wild-type animals exposed to preimmune IgG (P < 0.02, Wilcoxon stratified rank sum test). The apparent Vmax for ferrous iron uptake in HFE-deficient samples in the presence of antibody to DMT1 was not significantly different from control. Gastroenterology 2001 120, 1420-1429DOI: (10.1053/gast.2001.24050) Copyright © 2001 American Gastroenterological Association Terms and Conditions