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.

Separate pathways for cellular uptake of ferric and ferrous iron.

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.

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 beta(3) 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.

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.

Iron absorption and cellular transport: the mobilferrin/paraferritin paradigm.

Dietary inorganic iron is mostly ferric iron. This is solubilized at the acid pH level of the stomach where it chelates mucins and certain dietary constituents to keep them soluble and available for absorption in the more alkaline duodenum. Mucosal uptake of iron is facilitated by a beta 3 integrin and a 56 kDa protein known as mobilferrin. In the cytosol of the absorptive cell, iron is associated with a 520-kDa complex known as paraferritin which contains integrin, mobilferrin, and flavin monooxygenase. This complex serves as a ferrireductase to reduce iron to the ferrous state so that it is available for formation of end products such as heme proteins. The large complex has other constituents, such as beta 2 microglobulin, whose functions remain to be delineated. We postulate that the basolateral membranes of absorptive cells possess both holo-transferrin and apotransferrin receptors that regulate the ingress and egress of cellular iron, respectively. Unlike absorptive cells, nonintestinal cells appear to possess three pathways for uptake of inorganic iron: (1) the classical transferrin-transferrin receptor pathway, (2) the transferrin-associated transferrin receptor independent pathway (TRIP), and (3) the transferrin-independent mobilferrin-integrin pathway (MIP) observed in intestinal absorptive cells. The TRIP is used when transferrin receptors become saturated at physiological concentrations of iron and transferrin. The MIP may only be used efficiently for mucosal uptake of iron and iron-overloaded individuals with fully saturated transferrin. Alternatively, it may facilitate iron uptake from the TRIP after degradation of transferrin near the surface of the cell. However, both transferrin-associated pathways donate iron to a common intracellular iron pathway for ferri-reduction and probably other functions.

Absorption of heme iron.

Heme iron is absorbed from meat more efficiently than dietary inorganic iron and in a different manner. Thus, iron deficiency is less frequent in countries where meat constitutes a significant part of the diet. Proteolytic digestion of myoglobin and hemoglobin results in the release of heme, which is maintained in a soluble form by globin degradation products so that it remains available for absorption. Chelators that either diminish or enhance the absorption of inorganic iron have little effect on the absorption of heme iron. Heme enters the small intestinal absorptive cell as an intact metalloporphyrin. This may be facilitated by a vesicular transport system. In the absorptive cell the porphyrin ring is split by heme oxygenase. The released inorganic iron becomes associated with mobilferrin and paraferritin, which acts as a ferrireductase to make iron available for production of iron-containing end products such as heme proteins. Mucosal transfer of iron into the body occurs competitively with dietary iron that entered the absorptive cell as inorganic iron because they both share a common pathway within the intestinal cell.

The alternate iron transport pathway: mobilferrin and integrin in reticulocytes.

Iron transport in reticulocytes is known to occur via the well-described transferrin-receptor-endosome pathway. An alternative pathway for iron transport independent of transferrin has been postulated in reticulocytes and other cells. Transport of iron into reticulocytes from ferric citrate solutions was shown to be saturable and independent of transferrin. During transport of iron from ferric citrate, both cell surface integrins, and a soluble protein, mobilferrin, were labelled. This demonstrated that the reticulocyte transferrin independent pathway for iron transport involved integrins and mobilferrin similar to intestinal absorptive cells. This pathway would be expected to transport iron into cells under conditions of iron overload and was capable of providing iron for haemoglobin synthesis. Mobilferrin was also radiolabelled when radioiron labelled transferrin was incubated with reticulocytes and this occurred with a different time course than was observed following reticulocyte exposure to radiolabelled ferric citrate. This suggested that mobilferrin may serve as an intermediary in both pathways.

Mobilferrin is an intermediate in iron transport between transferrin and hemoglobin in K562 cells.

Iron is bound to transferrin in the plasma. A specific receptor on the cell surface binds transferrin and internalizes transferrin and the iron in clathrin-coated pits. These invaginate to form vesicles which release iron to the cytoplasm. Inorganic iron can be transported by an alternative pathway from iron citrate, utilizing a cell surface integrin and a cytoplasmic protein mobilferrin. This article shows that the two pathways donate iron to mobilferrin which acts as an intermediate between the iron bound to transferrin and the incorporation of iron into hemoglobin. Mobilferrin is found associated with the transferrin containing vesicles, and becomes labeled with iron released from transferrin in the vesicles. Mobilferrin is also found in the cytoplasm where pulse-chase experiments show that it, in turn, releases iron to be used for the synthesis of hemoglobin.

A phase II trial of piroxantrone in endometrial cancer: Southwest Oncology Group study 8918.

A phase II trial of the new anthrapyrazole piroxantrone was carried out by the Southwest Oncology Group in patients with advanced metastatic or recurrent endometrial cancer. A two-stage statistical design targeted accrual of 20 eligible patients. The starting dose of piroxantrone was 150 mg/m2 in patients without prior radiation therapy (RT) and 120 mg/m2 in patients with prior RT. There were 15 eligible patients, six of whom had received prior hormonal therapy while nine patients had not received prior hormonal therapy. Eight patients had received prior RT while seven patients had not received any prior RT. One to seven cycles of piroxantrone were administered. Dose escalation was feasible in four patients. No grade 5 toxicity was experienced by any patients. Most of the grade 4 (granulocytopenia in one) and grade 3 (leukopenia in three, granulocytopenia in three, anemia in two and thrombocytopenia in one) toxicity was related to myelosuppression. Grade 3 non-hematologic toxicities were nausea, fatigue and SGOT elevation. There was one partial response for a response rate of 7% (95% CI 0.2-32%) and median survival was 11 months (95% CI 3-13 months). The study was prematurely terminated due to lack of patient accrual.

Paraferritin: a protein complex with ferrireductase activity is associated with iron absorption in rats.

Recent studies reported that iron salts were absorbed in the duodenum utilizing a pathway involving membrane-associated integrin and a cytosolic protein named mobilferrin. In addition, a large molecular weight cytoplasmic complex was labeled with radioiron during mucosal uptake of iron in the duodenum. The molecular mass of this protein was 520 000 daltons, slightly larger than ferritin. On denaturing SDS-PAGE, the purified protein complex appeared to consist of at least four polypeptides, closely associated with each other. This complex was called paraferritin because its hydrodynamic volume resembled ferritin. In the present work, antibody studies demonstrate the presence of integrin, mobilferrin, and flavin monooxygenase in the water-soluble complex. Biochemical studies demonstrate the presence of a NADPH-dependent flavin monooxygenase ferrireductase activity that reduces Fe(III) to Fe(II). Antibodies against either integrin or mobilferrin inhibit monooxygenase activity. Inhibition of monooxygenase activity decreases radioiron uptake by tissue culture intestinal cells. Thus, we postulated that paraferritin plays a role in the mucosal uptake and transport of inorganic iron in small intestinal absorptive cells and is a mechanism for both the internalization of integrin from membranes to cellular cytosol and the delivery of iron to cellular constituents in an appropriate redox state.

Bone marrow necrosis.

Bone marrow necrosis is regarded as an uncommon entity that is associated with a poor prognosis. However, organized studies using either bone marrow biopsy specimens or autopsy material showed that bone marrow necrosis can be demonstrated in approximately one third of specimens. It is found in a large number of both malignant and nonmalignant disorders, in addition to occurring following large exposures to radiation or high dose cancer chemotherapy. In the absence of radiation or cancer chemotherapy, it probably eventuates from either vascular occlusion or blood stasis in small blood vessels. When bone marrow necrosis is prolonged, it may be associated with the development of bone marrow fibrosis, and it may serve as a predisposing etiology for idiopathic myelofibrosis. Most patients discovered with bone marrow necrosis have few symptoms, and they are eventually lost to follow-up without evident progression or development of a clinical illness. In acute disorders and in those who undergo effective therapy, recovery appears to occur without complications. This frequently overlooked finding is the subject of many case reports in the medical literature, but it has only been rarely systematically investigated. The latter is probably warranted because of the potential role of bone marrow necrosis in the pathophysiology of a number of disorders and the paucity of information for treatment of this pathological finding.

Hereditary hemochromatosis: a prevalent disorder of iron metabolism with an elusive etiology.

Hereditary hemochromatosis is a prevalent inherited disorder with an estimated frequency of homozygosity of 0.2 to 0.45% in Caucasians. The disease is characterized by progressive iron overload until a massive accumulation of body iron occurs. Undetected, the disorder eventually can produce either cirrhosis, diabetes mellitus, cardiac disease, arthritis, or hepatocellular carcinoma or a combination of these manifestations. Early diagnosis and treatment prevents organ damage and normalizes life expectancy. Screening studies to detect hemochromatosis are most effectively accomplished by measurement of the serum iron and total iron binding capacity. Treatment is most effectively performed by frequent phlebotomy until body stores are empty and then 3 to 4 times yearly for life. The basic defect of hemochromatosis appears to increase iron absorption, decrease iron excretion, and produce preferential deposit of iron in hepatic parenchymal cells rather than Kupffer cells. The genetic abnormality of hemochromatosis is located on chromosome 6 in close association with the gene for HLA antigens. Recent speculation postulates that tumor necrosis factor may be involved in the etiology of this disease because of its location on chromosome 6 and its effect upon iron transport.

Ironic catastrophes: one's food--another's poison.

Iron deficiency is an important nutritional problem in third world countries because it diminishes work performance. In meat-eating countries, iron excess may be more important than iron deficiency. Heme iron is more efficiently absorbed from the diet than inorganic iron, and iron excess can produce cellular oxidation in association with superoxide dismutase. Metal ion catalysis is linked to aging, coronary artery disease, stroke, carcinogenesis, neurodegenerative disorders, and inflammatory disorders. Prudence is advised in the excessive consumption of meat and iron supplementation of the diet until this process is more thoroughly investigated.