Marcel E. Conrad

DMT1: a mammalian transporter for multiple metals.

DMT1 has four names, transports as many as eight metals, may have four or more isoforms and carries out its transport for multiple purposes. This review is a start at sorting out these multiplicities. A G185R mutation results in diminished gastrointestinal iron uptake and decreased endosomal iron exit in microcytic mice and Belgrade rats. Comparison of mutant to normal rodents is one analytical tool. Ectopic expression is another. Antibodies that distinguish the isoforms are also useful. Two mRNA isoforms differ in the 3′ UTR: +IRE DMT1 has an IRE (Iron Responsive Element) but -IRE DMT1 lacks this feature. The ±IRE proteins differ in the distal 18 or 25 amino acid residues after shared identity for the proximal 543 residues. A major function is serving as the apical iron transporter in the lumen of the gut. The +IRE isoform appears to have that role. Another role is endosomal exit of iron. Some evidence indicts the -IRE isoform for this function. In our ectopic expression assay for metal uptake, four metals – Fe2+, Mn2+, Ni2+ and Co2+ – respond to the normal DMT1 cDNA but not the G185 R mutant. Two metals did not – Cd2+ and Zn2+ – and two – Cu2+ and Pb2+–remain to be tested. In competition experiments in the same assay, Cd2+, Cu2+ and Pb2+ inhibit Mn2+ uptake but Zn2+ did not. In rodent mutants, Fe and Mn appear more dependent on DMT1 than Cu and Zn. Experiments based on ectopic expression, specific antibodies that inhibit metal uptake and labeling data indicate that Fe3+ uptake depends on a different pathway in multiple cells. Two isoforms localize differently in a number of cell types. Unexpectedly, the -IRE isoform is in the nuclei of cells with neuronal properties. While the function of -IRE DMT1 in the nucleus is speculative, one may safely infer that this localization identifies new role(s) for this multifunctional transporter. Management of toxic challenges is another function related to metal homeostasis. Airways represent a gateway tissue for metal entry. Preliminary evidence using specific PCR primers and antibodies specific to the two isoforms indicates that -IRE mRNA and protein increase in response to exposure to metal in lungs and in a cell culture model; the +IRE form is unresponsive. Thus the -IRE form could be part of a detoxification system in which +IRE DMT1 does not participate. How does iron status affect other metals toxicity? In the case of Mn, iron deficiency may enhance cellular responses.

Iron absorption and transport—An update

Iron is vital for all living organisms. However, excess iron is hazardous because it produces free radical formation. Therefore, iron absorption is carefully regulated to maintain an equilibrium between absorption and body loss of iron. In countries where heme is a significant part of the diet, most body iron is derived from dietary heme iron because heme binds few of the luminal intestinal iron chelators that inhibit absorption of non‐heme iron. Uptake of luminal heme into enterocytes occurs as a metalloporphyrin. Intracellularly, iron is released from heme by heme oxygenase so that iron leaves the enterocyte to enter the plasma as non‐heme iron. Ferric iron is absorbed via a β3 integrin and mobilferrin (IMP) pathway that is not shared with other nutritional metals. Ferrous iron uptake is facilitated by DMT‐1 (Nramp‐2, DCT‐1) in a pathway shared with manganese. Other proteins were recently described which are believed to play a role in iron absorption. SFT (Stimulator of Iron Transport) is postulated to facilitate both ferric and ferrous iron uptake, and Hephaestin is thought to be important in transfer of iron from enterocytes into the plasma. The iron concentration within enterocytes reflects the total body iron and either upregulates or satiates iron‐binding sites on regulatory proteins. Enterocytes of hemochromatotics are iron‐depleted similarly to the absorptive cells of iron‐deficient subjects. Iron depletion, hemolysis, and hypoxia each can stimulate iron absorption. In non‐intestinal cells most iron uptake occurs via either the classical clathrin‐coated pathway utilizing transferrin receptors or the poorly defined transferrin receptor independent pathway. Non‐intestinal cells possess the IMP and DMT‐1 pathways though their role in the absence of iron overload is unclear. This suggests that these pathways have intracellular functions in addition to facilitating iron uptake. Am. J. Hematol. 64:287–298, 2000.

Iron absorption and transport.

Iron is vital for living organisms because it is essential for multiple metabolic processes to include oxygen transport, DNA synthesis, and electron transport. However, iron must be bound to proteins to prevent tissue damage from free radical formation. Thus, its concentrations in body organs must be regulated carefully. Intestinal absorption is the primary mechanism regulating iron concentrations in the body. Three pathways for intestinal iron uptake have been proposed and reported. These are the mobilferrin-integrin pathway, the divalent cation transporter 1 (DCT-1) [or natural resistance-associated macrophage protein (Nramp2)] pathway, and a separate pathway for uptake of heme by absorptive cells. Each of these pathways are incompletely described. However, studies with blocking antibodies, observations in rodents with disorders of iron metabolism, and studies in tissue culture cells suggest that the DCT-1 pathway is dominant in embryonic cells and is involved with cellular uptake of ferrous iron, whereas the mobilferrin-integrin pathway facilitates absorption of dietary inorganic ferric iron. Thus, there are separate pathways for cellular uptake of ferric and ferrous inorganic iron. Body iron can enter intestinal cells from plasma via basolateral membranes containing the classical transferrin receptor pathway with a high affinity for holotransferrin. This keeps the absorptive cell informed of the state of iron repletion of the host. Intestinal mucosal cell iron seems to exit the cell via a distinct apotransferrin receptor and a newly described protein named hephaestin. Unlike the absorptive surface of intestinal cells, most other cells possess transferrin receptors on their surfaces and the vast majority of iron entering these cells is transferrin associated. There seem to be 2 distinct pathways by which transferrin iron enters nonintestinal cells. In the classical clathrin-coated pitendosome pathway, iron accompanies transferrin into the cell to enter a vesicle, which releases the iron to the cytosol with acidification (high affinity, low capacity). Under physiological conditions, a second transferrin associated pathway (low affinity, high capacity) exists which has been named the transferrin receptor independent pathway (TRIP). How the TRIP delivers iron to cells is incompletely described. In addition, tissue culture studies show that nonintestinal cells can accept iron from soluble iron salts. This occurs via the mobilferrin-integrin and probably the DCT-1 pathways. Cellular uptake of iron from iron salts probably occurs in iron overloading disorders and may be responsible for free radical damage when the iron binding capacity of plasma is exceeded. Radioiron entering the cell via the heme and transferrin associated pathways can be found in isolates of mobilferrin/paraferritin and hemoglobin. This interaction probably occurs to permit NADPH dependent ferrireduction so iron can be used for synthesis of heme proteins. Production of heme from iron delivered via these routes indicates functional specificity for the pathways.

A role for mucin in the absorption of inorganic iron and other metal cations: A study in rats

The steps involved in iron absorption are poorly understood. Although transferrin and ferritin are water soluble, most radioiron in gut homogenates after an intraluminal dose of radioiron is recovered in water-insoluble precipitates. Most radioiron in the precipitates was insoluble in detergents and organic solvents and was characterized as mucins. These isolates bound iron in vitro with a Kd of 9.09 x 10(-5). Similar iron binding was observed with commercial mucins. Iron binding to mucin occurred at acid pH and maintained the iron available for absorption with alkalinization. Similar pH-dependent binding to mucin was observed with zinc, cobalt, and lead. Iron competitively inhibited binding of these metals to mucin. However, iron chelates of ascorbate, fructose, and histidine donated iron to mucin at neutral pH. These data provided a role for gastric HCl and intestinal mucin in absorption of iron and metal cations and partial explanation of the competition for absorption between certain metals from the gut lumen. It is postulated that intestinal mucin delivers inorganic iron to intestinal absorptive cells in an acceptable form for absorption.

Ultrastructural localization of transferrin, transferrin receptor, and iron‐binding sites on human placental and duodenal microvilli

Ultrastructural methods were used to determine the subcellular location of the transferrin receptor, transferrin and iron‐binding sites on human term placenta and human duodenum microvillus surfaces. The transferrin receptor and transferrin were localized by immunocytochemical methods employing either OKT9, a human transferrin receptor monoclonal antibody, or mouse anti‐human transferrin (ATfn), both followed by a horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG (GAM‐HRP) and diaminobenzidine (DAB) sequence. Iron‐binding sites were localized by acid ferrocyanide (AF) staining after saturation of tissue specimens with iron, accomplished with iron nitrilotriacetate (FeNTA), a known transferrin iron donor. Placental microvillus surfaces demonstrated staining for the OKT9‐GAM‐HRP‐DAB‐reactive transferrin receptor, ATfn‐GAM‐HRP‐DAB‐reactive transferrin, and FeNTA‐AF‐reactive iron acceptor, whereas enterocyte microvillus surfaces lacked significant staining with each of these methods. FeNTA‐AF stained iron‐binding substance in placental and enterocyte microvilli and cytoplasmic matrix. Thus using the same ultrastructural immunostaining and cytochemical methods transferrin receptor, transferrin, and nitrilotriacetate iron acceptor sites can be demonstrated on the microvillus surface of human placenta but not on the microvillus surface of human duodena.

A Phase II trial of epothilone B analogue BMS-247550 (NSC #710428) ixabepilone, in patients with advanced pancreas cancer: A Southwest Oncology Group study

SummaryPurpose: The purpose of this Phase II multi-institutional study was to define the efficacy and toxicity of ixabepilone in patients with advance pancreatic adenocarcinoma.n Patients and methods: Patients were required to have pancreatic adenocarcinoma and metastatic or recurrent disease that was not amenable to curative resection. Performance status was 0-1, and patients could not have had prior chemotherapy, or chemoradiation therapy for their advanced disease although prior local palliative radiation was allowed. Ixabepilone was administered iv as a 3xa0hour infusion every 21 days. Initially, the dose was 50xa0mg/m2 but this was lowered to 40xa0mg/m2 shortly after the trial opened because of concerns about neurotoxicity.n Results: Sixty-two patients were registered however 2 were ineligible because they did not have recurrent or metastatic disease. For the 60 eligible patients, 22 had performance status of 0 and 38 performance status of 1. The estimated 6-month survival was 60% (95% CI 48%–72%) with a median survival of 7.2 months and an estimated time to treatment failure of 2.3 months. Out of 56 patients with measurable disease there were 5 confirmed partial responses for a confirmed response probability of 9% (95% CI 3%–20%) and 7 unconfirmed partial responses for an overall response probability of 21% (95% CI 12%–34%). Common toxicities were neutropenia/granulocytopenia, nausea and vomiting and neuropathy. There was one death, cause not determined but judged “possibly” related to treatment.n Conclusion: Ixabepilone shows encouraging activity in patients with advanced pancreatic cancer and should be investigated further in this disease.

Rat duodenal iron-binding protein mobilferrin is a homologue of calreticulin.

BACKGROUNDnMobilferrin is a water soluble 56-kilodalton protein isolated from human and rat duodenal mucosa. It binds iron and other transitional metals in vivo and in vitro and is postulated to play a role in their absorption and intracellular metabolism. The purpose of this study was to characterize mobilferrin.nnnMETHODSnMobilferrin was characterized by identification of the N-terminal amino acid sequence, two-dimensional protein electrophoresis, and studies of mobilferrin and homologues using anti-mobilferrin antibody and competitive metal binding.nnnRESULTSnThe N-terminal amino acid sequence of mobilferrin was Asp-Pro-Ala-Ile-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-Ala-Ser-Thr- and is a homologue of calreticulin (calregulin). The proteins had a similar molecular mass (56 kilodalton) and isoelectric point (4.7). Anti-mobilferrin antibodies react with calreticulin. Both proteins bind iron and calcium but have a greater affinity for iron.nnnCONCLUSIONSnMobilferrin and calreticulin are homologues that bind iron with greater affinity than calcium and other transitional metals. Competitive binding of metals by mobilferrin provides insight into the absorptive pathway shared by both essential and toxic transitional metals.

Aplastic Crisis in Sickle Cell Disorders: Bone Marrow Necrosis and Human Parvovirus Infection

Aplastic crisis in patients with sickle cell disease who develop a parvovirus infection may be associated with extensive bone marrow necrosis as well as acute selective erythroblastopenia. This illness may be manifested by pyrexia, lymphadenopathy, bone tenderness and significant hypoxemia with minimal roentgenographic findings in the lungs. It is uncertain whether the hypoxemia is caused by the effects of the viral infection on the lungs or is secondary to sickling of red blood cells in the pulmonary vasculature or both. The hypoxia may be sufficiently severe to require treatment with both oxygen and transfusion. The physical damage to the bone marrow associated with bone marrow necrosis may be more important than selective acute erythroblastopenia in inducing aplastic crisis in patients with sickle cell disorders. Studies of bone marrow biopsy specimens collected during parvovirus-associated aplastic crisis in patients with nonsickle cell hemolytic disorders would be helpful in determining the pathophysiology of parvovirus-associated disorders.

Iron Absorption and Cellular Uptake of Iron

Iron balance is regulated primarily by keeping the absorptive process attuned to body requirements. While iron loss from the body is quantitatively as important as iron absorption, excretion is limited and plays a more passive role [1]. It has been known for many years that iron is absorbed in the small intestine. However, the mechanisms involved in mucosal uptake of dietary iron and mucosal transfer of iron into the plasma are poorly understood. In non-intestinal cells which possess transferrin receptors on their surface, iron is believed to enter the cell via a transferrin, transferrin-receptor clathrin mediated mechanism [2]. However, recent publications suggest that there may be an alternative pathway [3-5].

Abnormalities of flavin monooxygenase as an etiology for sideroblastic anemia

We postulated that a deficiency of flavin monooxygenase (FMO)—a ferrireductase component of cells—could produce sideroblastic anemia. FMO is an intracellular ferrireductase which may be responsible for the obligatory reduction of ferric to ferrous iron so that reduced iron can be incorporated into heme by ferrochelatase. Abnormalities of this mechanism could result in accumulation of excess ferric iron in mitochondria of erythroid cells to produce ringed sideroblasts and impair hemoglobin synthesis. To investigate this hypothesis we obtained blood from patients with sideroblastic anemia and normal subjects. Extracts of peripheral blood lymphocytes were used to measure ferrireduction by utilization of NADPH. Lymphoid precursors are reported to accumulate iron in mitochondria similarly to erythroid precursors. Utilization of lymphoid precursors avoided the need for bone marrow aspirations. We studied three patients with sideroblastic anemia. One patient and his asymptomatic daughter had a significant decrease in ferrireductase activity. They also had markedly diminished concentrations of FMO in lymphocyte protein extracts on Western blots. This was accompanied by increased concentration of mobilferrin in the extracts. These results suggest that abnormalities of FMO and mobilferrin may cause sideroblastic anemia and erythropoietic hemochromatosis in some patients. Am. J. Hematol. 65:149–153, 2000.