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The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.

Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.

Intestinal nonheme iron levels and mRNA levels of genes implicated in iron metabolism were measured in mice with altered iron metabolism [chronic (4 wk) and acute (4 days) dietary iron deficiency; iron overload and hypoxia] to investigate their role in the process and regulation of intestinal iron absorption. Mucosal nonheme iron levels were decreased by both chronic and acute iron deficiency and increased by iron overload but were not affected by hypoxia. There was evidence of a gradient of mucosal nonheme iron along the small intestine (duodenum, jejunum > ileum). There were also regional differences in H-ferritin (duodenum > ileum) and transferrin receptor (ileum > duodenum) mRNA levels. Iron overload produced a decrease in transferrin receptor (TfR) mRNA in the duodenum, with ferritin mRNA levels unaffected in both the duodenum and ileum. Chronic iron deficiency induced a twofold increase in TfR mRNA levels in both the duodenum and ileum, whereas H- and L-ferritin mRNA levels did not change significantly. The ratio of H- to L-ferritin mRNA decreased significantly during exposure to hypoxia; however, individual ferritin and TfR mRNA levels were not significantly altered. Calreticulin (mobilferrin), cysteine-rich intestinal protein, and H(+)-adenosinetriphosphatase mRNA levels were virtually unchanged in all models. A comparison with previously published data on changes in iron absorption leads us to conclude that 1) iron absorption can be altered independently of effects on transcripts of genes for iron-related proteins, and 2) it is not essential for iron absorption to be coordinated with regulation of mucosal iron metabolism.

Background: Previous in vitro studies have shown that the uptake of Fe(III) by freshly isolated duodenal mucosal biopsy specimens is increased in patients with genetic haemochromatosis. Moreover, in the mouse it has recently been found that reduction of Fe(III) to Fe(II) is a prerequisite for iron uptake by the proximal intestine., Aims/methods: This study used the in vitro technique to investigate the rates of reduction and uptake of 59Fe(III) by duodenal mucosal biopsy specimens obtained at endoscopy from treated and untreated patients with genetic haemochromatosis., Results: The rate of reduction of iron in the medium was proportional to the incubation time and was not caused by the release of reducing factors from the tissue fragments. Ferrozine, a specific Fe(II) chelator and ferricyanide, a non-permeable oxidising agent, inhibited uptake of 59Fe showing that reduction of Fe(III) precedes uptake. The rates (all values given as pmol/mg/min) of reduction (152 (49) v 92 (23)) and uptake (8.3 (4.0) v 3.6 (1.3), mean (SD)), were significantly increased in biopsy specimens from the untreated group (n = 6) compared with those from 10 control subjects (p < 0.04). Furthermore, the reduction and uptake rates were still increased in five patients in whom iron stores were normal after venesection treatment., Conclusions: These results show that there is a persistent abnormality in the reduction and uptake of iron by the intestine in genetic haemochromatosis.

Mouse duodenum possesses mucosal surface ferricyanide reductase activity. The reducing activity, determined in vitro by measuring ferrocyanide production from ferricyanide, was found to be greater in duodenal fragments when compared with ileal fragments. Experiments with right-side out tied-off duodenal sacs show that reduction occurs mainly on the mucosal side and indicates that the reducing activity is associated with the brush border membrane. Experiments using mice with increased levels of iron absorption (hypoxic, iron-deficient) showed corresponding increases in reducing activity. The increase was present in duodenal but not ileal fragments. Inhibitor studies showed no effect of several compounds which inhibit other, more characterized, transplasma membrane reductases. In particular, doxorubicin (10 microM) and quinacrine (1mM) were without effect on duodenal mucosal transplasma membrane reducing activity. Depolarization of the membrane potential with high medium K+ inhibited reducing activity. N-ethyl malemide (1 mM) was a potent inhibitor, but iodoacetate was found to be less inhibitory. Comparison with inhibitory effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated that the effect of N-ethyl malemide on reducing activity was not secondary to GAPDH. Collectively these results indicate that mouse duodenum possesses mucosal surface transplasma membrane ferricyanide reductase activity and that the activity is correlated with the process of intestinal iron absorption. Furthermore, the reducing activity appears to be distinct from other reported transplasma membrane reductases.

Cytosolic aconitase activity was assayed in duodenal mucosa from mice subjected to a variety of manipulations known to modulate duodenal iron status and duodenal iron absorption. No changes in cytosolic aconitase activity were observed 1 h after oral FeSO4 dosing or intramuscular desferrioxamine treatment. Three days of hypoxic exposure and two weeks treatment with intramuscular iron dextran also had no effect on cytosolic aconitase. Three weeks growth on an iron deficient diet significantly reduced cytosolic aconitase activity. In no situation was there any evidence for significant amounts of inactive aconitase which could be activated in vitro with FeSO4/cysteine. These data suggest that duodenal cytosolic aconitase is not sensitive to acute changes in mucosal iron levels and is generally much less sensitive to body iron status than is duodenal iron absorption. There is evidence that chronic iron depletion reduces cytosolic aconitase to a relatively small degree but generally activity is maintained, consistent with an important metabolic role for the enzyme.

Duodenal biopsies from control subjects, patients with iron-deficiency anaemia and rheumatoid arthritic patients with anaemia of chronic disorders (ACD) were investigated for their ability to take up 59Fe from iron ascorbate. Additionally, duodenal tissues were analysed for iron and immunoreactive ferritin and transferrin. Biopsies from iron-deficient subjects showed a 2- to 3-fold increase in the apparent Vmax for 59Fe uptake, compared to control values. Uptake was inversely related to body iron stores. ACD patients showed similar rates of 59Fe uptake to controls; the rates were independent of the degree of anaemia or serum ferritin levels. Tissue analysis showed reductions in mucosal iron and ferritin levels in iron-deficient patients, whilst transferrin levels were within the normal range. ACD patients also exhibited lower mucosal iron levels, but had iron protein levels within the normal range. It is suggested that factors distant from the intestinal mucosa influence iron absorption in ACD.

Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (M(r) approximately 18,000, pI 6.7-6.9) and b (M(r) approximately 24,000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (M(r) approximately 66,000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.

Partition and distribution coefficients (log P, log D) into rat small intestinal brush border membrane (BBM) were measured for a variety of ionizable and non-ionizable drugs and solutes using a novel technique. The log P values were compared with those determined with model solvents, octanol and propylene glycol dipelargonate (PGDP). Non-ionizable solutes with log P values up to 3.0 showed that octanol was a better model for partition into the BBM than PGDP. With one exception, BBM partition coefficients of greater than 3 were not observed, even for solutes with log P values in model solvents that were greater than 5. Liposomes prepared from BBM lipids, or synthetic lipid mixtures of similar composition to BBM, demonstrated similar trends in partition coefficients to the intact BBM. Two cationic drugs, Atenolol and Xamoterol were investigated for partition into BBM lipid liposomes. An apparent enhancement of log D with respect to octanol was attributed to a "surfactant-like" orientation in the membrane and an interaction of the ionized drug with anionic phospholipid head groups. The anionic drug Proxicromil shows the expected decrease in log D with increasing pH, at low NaCl concentrations. Changes in electrophoretic mobility of liposomes after incorporation of Proxicromil into them were consistent with the negative charge of the ionized drug being at the membrane surface. It was concluded that Proxicromil also associates with membranes in a "surfactant-like" orientation and that increased extraction with increasing NaCl concentrations is a result of ionic strength effects. Partition of solutes into BBM vesicles is more complex than into organic solvents and probably represents an important step in overall intestinal permeation of solutes.

Colonic growth factors (CGFs) were extracted from porcine intestinal epithelium and mucosa. Under acidic conditions, very little mitogenic activity (as assayed using murine 3T3 fibroblasts and a human colonic cell line) was extractable. However, by extracting at neutral or slightly alkaline pH, significant mitogenic activity for both the murine fibroblasts and human colonic carcinoma cell line could be detected. CGFs are present throughout the intestine and cecum. The epithelial mucosa of the distal colorectal region appeared to contain mitogens which were more potent for the colonic cells than the 3T3 fibroblasts. Purification of CGFs from the colonic mucosa required removal of associated mucin by pH precipitation prior to chromatographic fractionation. It was then possible to develop a complete purification (390,000-fold) scheme for the major CGF, an 18-kDa protein which bound to heparin-Sepharose. N-terminal sequence analysis yielded a single sequence (Q)SPGGAMAAGSITTLPALP, i.e. an N-terminally extended form of basic fibroblast growth factor. Apart from the substitution of Gly in bovine basic fibroblast growth factor by a Ser in porcine CGF, the proteins are identical. A similar extraction procedure using purified human colonic crypt epithelial cells yielded a mitogen for the human colonic cell line with similar chromatographic properties.

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.

Three days hypoxia (0.5 atm) increased the haemoglobin and haematocrit values in rats paralleled by enhanced intestinal iron absorption. The destination of recently-absorbed iron was primarily the erythropoietic system, viz. bone marrow, spleen and red cells. Total plasma transferrin, was increased by 30%, but no significant changes in mucosal transferrin were found. No increase in labelling of mucosal transferrin by absorbed iron was observed. These results suggest that mucosal transferrin does not play a major role in the regulation of intestinal iron absorption in hypoxia.

Mouse intestinal brush-border membrane vesicles take up iron from media containing 59Fe3 +-nitrilotriacetic acid. The iron uptake by the vesicles represents accumulation of iron which relates to an osmotically active space. Uptake is linearly related to vesicle protein concentration and is inhibited by low incubation temperature and low medium free Fe3+ concentrations. Experiments with the lipid soluble iron ligand 8-hydroxyquinoline and with Triton X-100 imply that the uptake is rate limited by membrane transport.

Initial rates of mucosal uptake of Fe3+ from luminal Fe3+-nitrilotriacetate solutions by tied segments of mouse intestine in vivo have been measured. Duodenal uptake showed an approximately hyperbolic dependence of uptake on Fe3+ complex concentration (Km(app) 66 microM, Vmax 6.2 pmol/min per mg intestine) with little dependence on nitrilotriacetate:Fe3+ ratio or on added Ca2+. Duodenal uptake was greatly stimulated by hypoxic treatment of mice. Uptake rates by distal ileum were lower than by duodenum and more sensitive to added Ca2+. These results show that isolated duodenal brush-border membrane Fe3+ transport characteristics (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226) are inadequate to explain duodenal Fe3+ uptake in vivo. However, ileal uptake can be explained by the properties of isolated ileal brush-border membrane (Simpson, R.J., Raja, K.B. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 8-12).

In order to define the importance of the mucosal uptake step in the intestinal regulation of iron absorption, unidirectional uptake rates of Fe3+ from a nitrilotriacetic acid chelate were measured in duodenal fragments from mice using an in vitro technique. [57Co]-Cyanocobalamin was used as a marker of adherent incubation medium. Uptake showed saturation kinetics over the concentration range 18-450 microM. Uptake was increased in fragments from hypoxic, dietary iron-deficient and pregnant mice. The enhanced uptake was due to an increase in Vmaxapp. However, the modest increase in uptake rates in pregnancy and the gross changes observed in iron-deficiency make the hypoxic model the most convenient. The increase in uptake in hypoxic animals was located to the duodenal region and was not associated with changes in either total mucosal iron content or epithelial cell turnover. The rate of uptake of iron via the serosa did not change with hypoxia. This study implies that flux of Fe3+ across the brush border is subject to adaptive regulation. The hypoxic model is suitable for investigation into the regulation of iron homeostasis.

A successful method for the analytical subcellular fractionation of mouse duodenal mucosa organelles was established. 59Fe(III)-nitrilotriacetate (pH 7.2) was injected into tied-off duodenal segments in vivo and, after 2-20 min, mucosal homogenates were subjected to subcellular fractionation. Radioactivity was recovered in the cytosolic fractions and in the gradient at a density of 1.18-1.20 g/ml. Enhanced iron absorption was achieved by placing the animals in a hypobaric chamber for 3 days. These animals had a higher proportion of particulate 59Fe compared to controls. Homogenisation in sucrose medium containing the selective plasma membrane perturbant digitonin shifted the particulate iron fraction to a higher density region of the gradient indicating a localisation of the iron binding site to the plasma membrane region of the mucosal cells. No significant radioactive iron was observed in the brush-border region of the gradient. Transferrin immunoreactivity was found only in the cytosolic region of the gradient and was not associated with any organelle.

Initial Fe3+ uptake rates by mouse intestinal fragments were determined in vitro. Uptake was dependent primarily on the Fe3+-nitrilotriacetate complex concentration. Addition of Ca2+ and Mg2+ to the incubation medium had only small effects on the Fe3+ uptake rate. Duodenal fragments from hypoxic animals showed enhanced uptake of Fe3+; this increase was more pronounced with a divalent cation-free medium. Ca2+ markedly diminished the Fe3+ uptake by mucosa from hypoxic mice; Mg2+ had no appreciable effect. Distal ileal fragments exhibited lower uptake rates compared to the duodenum, but were more sensitive to the effects of added Ca2+. The ileal fragments did not show an adaptive response of Fe3+ uptake to hypoxia. These results suggest the existence of more than one pathway for mucosal Fe3+ uptake. One pathway, sensitive to Ca2+ and not stimulated by hypoxia, may be present in the duodenum and ileum. A second pathway, inhibited by Ca2+ and exhibiting an adaptive response to hypoxia, occurs only in the duodenum. This latter pathway is more sensitive to the effects of metabolic inhibitors.

Recent studies of Fe2+ uptake by mouse proximal intestine brush-border membrane vesicles revealed low-affinity, NaCl-sensitive and high-affinity, NaCl-insensitive, components of uptake (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 381-388). In this study, the former component is demonstrated to show a strong pH dependence with an optimum of pH 6.8-6.9. Studies at pH 6.5, where the low affinity component is inhibited by more than 25-fold compared with pH 7.2, suggest that the pH-sensitive component represents transport across the brush-border membrane followed by intravesicular binding. Cholate extracts of brush-border membrane vesicles contain pH- and NaCl-sensitive Fe2+ binding moieties which may be involved in the transfer of Fe2+ across the intestinal brush-border membrane and subsequent binding inside the vesicles. Fe2+ uptake by brush-border membrane vesicles from the duodenum of hypoxic mice is higher than uptake by vesicles from control-fed animals, suggesting the existence of a regulable brush-border membrane Fe2+ carrier.

Brush border membrane vesicles prepared using divalent cation precipitation methods can contain unphysiological levels of non-esterified fatty acids. Fatty acid production from endogenous lipid during brush border membrane vesicle preparation is effectively prevented by the lipase inhibitor diethyl 4-nitrophenylphosphate plus cooling. Vesicles prepared using this procedure have variable levels of non-esterified fatty acids (range 22-193 nmol mg-1 protein). Changes in non-esterified fatty acid levels in brush border membrane vesicles parallel Fe uptake by vesicles from Fe/ascorbate solutions. Brush border membrane vesicle fatty acids appear to be derived from the diet but hypoxic mice are able to maintain high brush border membrane non-esterified fatty acid levels despite reduced dietary intake. Non-esterified fatty acids in brush border membrane may thus provide a physiological mechanism of mucosal Fe uptake.

In vivo kinetics of mucosal uptake of luminal 59Fe2+ by tied segments of normal mouse duodenum are characterised by a Km of approx. 100 microM and a Vmax of approx. 9 pmol/min per mg wet weight of intestine. These values were determined at pH 7.25 in the presence of excess sodium ascorbate. Studies with luminal Fe2+ concentrations of 100 microM reveal: uptake is relatively independent of ascorbate: Fe ratio and luminal pH and uptake is potently inhibited by 1 mM Co2+ or Mn2+ and large luminal NaCl concentrations but not by Ca2+. 3 days of hypoxia (0.5 atmospheres) yields no significant increase in subsequent total mucosal uptake by in vivo tied segments while uptake is significantly reduced by semi-starvation. Quantitative comparison of in vivo mucosal uptake with subsequent determination of isolated brush-border membrane 59Fe2+ transport in individual mice reveals a positive correlation (P less than 0.01) between the two parameters. These results, in conjunction with studies of isolated mouse duodenal brush-border membrane (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta, 814, 381-388 and (1986) Biochim. Biophys. Acta 856, 109-114) suggest that the Fe2+ transport properties of isolated brush-border membrane are quantitatively adequate to explain in vivo mucosal uptake in normal and hypoxic mice at Fe2+ concentrations up to 100 microM.

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.

Intestinal iron uptake by mouse duodenal fragments is inhibited in the absence of oxygen and glucose from the incubation medium and by a variety of metabolic inhibitors. The mechanism of energy coupling to iron uptake is, however, unclear. In vitro experiments using duodenal fragments showed Fe3+ uptake to be markedly inhibited, in a reversible fashion, by the replacement of incubation medium Na+ by K+. Addition of phloridzin to the medium failed to affect iron uptake, suggesting that the above effect was not a consequence of reduced glucose uptake. Substitution of Na+ by Rb+ also potently reduced duodenal iron uptake. Replacement of medium NaCl by either mannitol or choline chloride had no significant effect on Fe3+ uptake, thus excluding the possibility of the Fe3+ uptake process being Na+-dependent. Similar observations were made with duodenal fragments from animals with enhanced Fe3+ absorption, due to chronic hypoxia. Valinomycin (1-5 microM) increased the uptake of both glucose and Fe3+. Higher concentrations (22.5 microM) of the ionophore were inhibitory. In vivo studies (tied-off segments) using Rb+-containing medium confirmed the inhibitory effects of univalent cations on Fe3+ absorption. Enhanced absorption of Fe3+ was also demonstrable in vivo, with low concentrations of valinomycin and nigericin added to the luminal medium. These observations suggest that the Fe3+ uptake process may be dependent on the brush-border membrane potential.