Biochemistry of nonheme iron in man. II. Absorption of iron

The currently accepted concept of iron absorption proposes first the entry of iron into the intestinal mucosal cell through the brush border membrane. It is a relatively slow process. In the cell, the iron may be transferred to plasma or become sequestered by ferritin. The latter becomes unavailable for transfer to plasma and is exfoliated and excreted. In iron deficiency and idiopathic hemochromatosis, the rate of iron uptake into the intestinal mucosal cell is increased and entry into ferritin is decreased, whereas the rate of transfer to plasma remains constant. The reverse occurs in case of secondary iron overload. It is currently accepted that a transferrin, whose levels increase in iron deficiency, enters the intestinal lumen from the liver via bile, where it may sequester iron and bring it into the cells by the process of endocytosis. Iron presented as inorganic ferric or ferrous salts may also be absorbed, though the more soluble ferrous salts are adsorbed much more rapidly. Heme iron is absorbed very effectively, though it is not subject to regulation by the individual's iron status to the same extent as is inorganic iron absorption. Brush border membranes apparently contain saturable iron receptors for inorganic iron, but whether or not the absorption process requires energy is an open question. Absorption of iron may also be affected by its availability; different food components affect iron absorbability to a different extent.     

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

Relationships between the copper and iron systems in hemodialysis patients and variables affecting these systems

The copper-binding protein ceruloplasmin oxidizes ferrous iron to ferric iron, an action that is critical for the binding of iron to transferrin in plasma. Ceruloplasmin, in common with ferritin and transferrin, is an acute-phase protein that is altered by inflammation. We sought to identify interrelationships between the copper and iron systems by measuring copper, ceruloplasmin, ferroxidase, ferritin, transferrin, iron, and iron-binding capacity in a group of hemodialysis patients. We looked for evidence of inflammation and free-radical injury by assaying for protein carbonyl groups, protein pyrrolation, di-tyrosine, and advanced oxidation protein products. Our findings were compatible with an active inflammatory state that affected both iron and copper metabolism. Transferrin levels were low, whereas ceruloplasmin levels were elevated compared to normal. Copper concentration was increased proportional to ceruloplasmin. Several variables including ceruloplasmin and transferrin were observed to correlate significantly with the level of pyrrolated protein. The data suggest that posttranslational modification of circulating proteins may affect their structural, enzymatic, and ligand-binding properties. Abnormalities in copper metabolism and their influence on iron handling in renal failure are complex and will require additional study before their importance can be defined.     

Transferrin receptor 2: a new molecule in iron metabolism

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

Intracellular processing of transferrin and iron by isolated rat hepatocytes.

Transferrin bound by isolated rat hepatocytes is rapidly endocytosed and enters a compartment of low density. Little was found associated with the lysosomes, even though the protein was subsequently lost from the cells. Iron entering the cells on transferrin was subsequently found in a number of intracellular components: transferrin, haem, ferritin and a residual fraction. After 2 h incubation with 59Fe-transferrin almost 70% of the iron was in ferritin, and this proportion increased to 80% during a 'chase' experiment. Residual iron, because of its rapid increase at the start of the incubation and its decline during the 'chase', probably represents an intracellular transit pool, which at steady state was present at 23 pg/10(6) cells.     

Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.     

Brain iron homeostasis.

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondrial respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.     

A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation

Of the different growth supplements used in chemically defined media, only transferrin is required for differentiation of tubules in the embryonic mouse metanephros. Since transferrin is an iron-carrying protein, we asked whether iron is crucial for tubulogenesis. Differentiation of metanephric tubules both in whole embryonic kidneys and in a transfilter system was studied. The tissues were grown in chemically defined media containing transferrin, apotransferrin, the metal-chelator complex ferric pyridoxal isonicotinoyl hydrazone (FePIH), and excesses of ferric ion. Although we found that apotransferrin was not as effective as iron-loaded transferrin in promoting proliferation in the differentiating kidneys, excess ferric ion at up to 100 microM, five times the normal serum concentration, could not promote differentiation or proliferation. However, iron coupled to the nonphysiological, lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone, to form FePIH, could sustain levels of cell proliferation and tubulogenesis similar to those attained by transferrin. Thus, the role of transferrin in cell proliferation during tubulogenesis is solely to provide iron. Since FePIH apparently bypasses the receptor-mediated route of iron intake, the use of FePIH as a tool for investigating cell proliferation and its regulation is suggested.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

The formation of ferritin from apoferritin. Catalytic action of apoferritin

The iron-storage protein ferritin consists of a protein shell and has an iron content of up to 4500 iron atoms as a microcrystalline ferric oxide hydrate. A study was made of the uptake of ferrous iron by apoferritin in the presence of an oxidizing agent at very low iron:protein ratios. At ratios of less than about 150 iron atoms per apoferritin molecule hyperbolic progress curves were obtained, whereas at higher ratios the curves became sigmoidal under the conditions used. A computer model, developed previously (Macara et al., 1972), was shown to account for this result. The experimental evidence indicates that apoferritin binds ferrous iron and catalyses the initial stage in the formation of the ferric oxide hydrate inside the protein shell. This stage involves the oxidation of sufficient iron within the protein molecule to form a stable nucleus on which the growth of the microcrystalline iron-core particles can proceed. A possible schematic mechanism for the action of apoferritin is suggested.     

Removal of ferritin-bound iron by DL-dihydrolipoate and DL-dihydrolipoamide

The naturally occurring dithiols DL-dihydrolipoate and DL-dihydrolipoamide were tested for their ability in the removal of ferritin-bound iron. Both compounds remove the iron stored inside the protein by complexing it in the ferric form. The iron can be reduced to the ferrous form by excess dithiol, but this is not necessary for complete removal. Reaction is complete in few hours and, at molar ratios of chelator to metal higher than 10, more than 60% of the ferritin-bound iron was removed. The amount of iron stored in the ferritin molecule does not affect the rate and the yield of the removal reaction. The iron-removing ability of DL-dihydrolipoate was found to be identical to that of an equimolar solution of sodium dithionite, and to be pH-dependent. Results are discussed in terms of the molecular architecture of ferritin and of the chelators, and their possible physiological relevance is pointed out.     

The cellular mechanisms of body iron homeostasis

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.     

Uptake of iron from transferrin by isolated rat hepatocytes. A redox-mediated plasma membrane process?

The uptake of iron from transferrin by isolated rat hepatocytes varies in parallel with plasma membrane NADH:ferricyanide oxidoreductase activity, is inhibited by ferricyanide, ferric, and ferrous iron chelators, divalent transition metal cations, and depends on calcium ions. Iron uptake does not depend on endosomal acidification or endocytosis of transferrin. The results are compatible with a model in which iron, at transferrin concentrations above that needed to saturate the transferrin receptor, is taken up from transferrin predominantly by mechanisms located to or contiguous with the plasma membrane. The process involves labilization and reduction of transferrin-bound iron by cooperative proton and electron fluxes. A model which combines the plasma membrane mechanism and the receptor-mediated endocytosis mechanism is presented.     

The pivotal role of ferritin in cellular iron homeostasis

Iron delivered by transferrin to the interior of the cell is in part utilized in biosynthetic processes and in part incorporated into ferritin, the major iron storage protein. The intracellular ferritin concentration is directly correlated to and determined by the extent of iron supply to the cell. Intracellular partitioning of iron to ferritin is suggested as forming the basis of cellular iron homeostasis.     

胎盘铁转运蛋白Zyklopen研究进展

Zyklopen（ZP）作为铜蓝蛋白的同系物,是近年来发现的铁转运蛋白.ZP具有一个位于羧基末端的跨膜结构域,在胎盘大量表达,也分布于脑、肾、视网膜、乳腺、睾丸等组织,但目前还不清楚ZP在这些组织中的功能.ZP具有亚铁氧化酶的活性,细胞内缺铜会引起ZP蛋白表达减少.在胎盘中,ZP可能通过将二价铁离子氧化为三价铁离子,帮助三价铁与胎儿循环系统中的转铁蛋白相结合,从而参与铁从母体到胎儿的转运过程.     

Computational Modeling and Analysis of Iron Release from Macrophages

A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe²⁺) through ferroportin (FPN), the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp), oxidizes ferrous to ferric ion. Apo-transferrin (Tf), the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can correctly predict cellular iron efflux and is essential for physiologically relevant whole-body model of iron metabolism.     

The formation of ferritin from apoferritin. Kinetics and mechanism of iron uptake

Ferritin has a high capacity as an iron store, incorporating some 4500 iron atoms as a microcrystalline ferric oxide hydrate. Starting from apoferritin, or ferritin of low iron content, Fe²⁺ and an oxidizing agent, the uptake of iron can be recorded spectrophotometrically. Progress curves were obtained and the reconstituted ferritin was shown by several physical methods to be similar to natural ferritin. The progress curves of iron uptake by apoferritin are sigmoidal; those for ferritins of low iron content are hyperbolic. The rate of iron uptake is dependent on the amount of iron already present in the molecule. The distribution of iron contents among reconstituted ferritin molecules is inhomogeneous. These findings are interpreted in terms of a crystal growth model. The surface area of the crystallites forming inside the protein increases until the molecule is half full, and then declines. This surface controls the rate at which new material is deposited. The experimental results can best be accounted for by a two-stage mechanism, an initial slow `nucleation' stage, which is apparently zero order with respect to [Fe<sup>2+</sup>], followed by a more rapid `growth' stage. The rate of Fe²⁺ oxidation is increased in the presence of apoferritin as compared with controls. Ferritin can therefore be regarded as an enzyme to which the product remains firmly attached. The protein appears to increase the rate of `nucleation'. The apparent zero order of this stage suggests the presence of binding sites on the protein, which are saturated with respect to Fe²⁺. These sites are presumed also to be oxidation sites. The oxidation and subsequent formation of the ferric oxide hydrate may proceed according to one of three alternative models.