Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.     

The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell.

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.     

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

Mucosal iron binding proteins and the inhibition of iron absorption by endotoxin

The uptake of iron from a tied off jejunal segment into the body after the injection of a 59Fe labeled test dose was decreased after the administration of endotoxin by about 80% in both normal and iron deficient animals.--In the iron deficient group the distribution of 59Fe in the cytosol fraction of jejunal mucosa between transferrin and ferritin was determined chromatographically; the amount of 59Fe in the ferritin fraction increased remarkably after the endotoxin treatment and the ratio of both was changed in favor of ferritin.--It is hypothesized that the association of the diversion of iron to the mucosal ferritin with the decrease of the transport of iron into the blood caused by endotoxin might be the consequence of abnormal oxidations in the mucosa measured by others in liver tissue.     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Ferritin iron kinetics and protein turnover in K562 cells

The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes.     

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.     

Factors involved in the regulation of iron transport through reticuloendothelial cells

The effects of various maneuvers on the handling of 59Fe-labeled heat-damaged red cells (59Fe HDRC) by the reticuloendothelial system were studied in rats. Raising the saturation of transferrin with oral carbonyl iron had little effect on splenic release of 59Fe but markedly inhibited hepatic release. Splenic 59Fe release was, however, inhibited by the prior administration of unlabeled HDRC or by the combination of carbonyl iron and unlabeled HDRC. When carbonyl iron was administered with unlabeled free hemoglobin, the pattern of 59Fe distribution was the same as that observed when carbonyl iron was given alone. 59Fe ferritin was identified in the serum after the administration of 59Fe HDRC but the size of the fraction was not affected by raising the saturation of transferrin. Sizing column analyses of tissue extracts from the spleen at various times after the administration of 59Fe HDRC revealed a progressive shift from hemoglobin to ferritin, with only small amounts present in a small molecular weight fraction. The small molecular weight fraction was greater in hepatic extracts, with the difference being marked in animals that had received prior carbonyl iron. The increased hepatic retention of 59Fe associated with a raised saturation of transferrin was reduced by a hydrophobic ferrous chelator (2,2'-bipyridine), a hydrophilic ferric chelator (desferrioxamine), and an extracellular hydrophilic ferric chelator (diethylene-triaminepentacetic acid). Transmembrane iron transport did not seem to be a rate-limiting factor in iron release, since no differences in 59Fe membrane fractions were noted in the different experimental settings. These findings are consistent with a model in which RE cells release iron from catabolized red cells at a relatively constant rate. When the saturation of transferrin is raised, a significant proportion of the iron is transported from the spleen to the liver either in small molecular weight complexes or in ferritin. Although a saturated transferrin had no effect on the release of iron from reticuloendothelial cells, prior loading with HDRC conditions them to release less iron.     

Transferrin and iron uptake by rat reticulocytes

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.     

Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.

The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.     

Modulation of cellular iron metabolism by hydrogen peroxide. Effects of H2O2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts

Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H(2)O(2) that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 microm H(2)O(2) for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H(2)O(2) induces a approximately 4-fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H(2)O(2)-treated cells display an increased capacity to sequester (59)Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of (59)Fe intracellular distribution. Our data suggest that H(2)O(2) regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms.     

Regulation of intracellular iron distribution in K562 human erythroleukemia cells

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.     

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Summary A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ^–1 and with 6.6×10^–12 moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ^–1 and 1.0×10^–12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Subcellular localization of ferritin and iron taken up by rat hepatocytes.

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.     

Influence of iron-saturation of plasma transferrin in iron distribution in the brain

Based on the evidence that iron distribution in the peripheral tissues is changed by iron-saturation of plasma transferrin, the influence of iron-saturation of plasma transferrin in iron delivery to the brain was examined. Mouse plasma was pre-incubated with ferric chloride in citrate buffer to saturate transferrin and then incubated with (59)FeCl(3). Peak retention time of (59)Fe was transferred from the retention time of transferrin to that of mercaptalbumin, suggesting that iron may bind to albumin in the plasma in the case of iron-saturation of transferrin. When mice were intravenously injected with ferric chloride in citrate buffer 10 min before intravenous injection of (59)FeCl(3), 59Fe concentration in the plasma was remarkably low. (59)Fe concentration in the liver of iron-loaded mice was four times higher than in control, while 59Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of (59)FeCl(3), brain autoradiograms also showed that (59)Fe concentrations in the brain of iron-loaded mice were approximately 40-50% of those of control mice in all brain regions tested except the choroid plexus, in which (59)Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain.     

Uptake and subcellular distribution of injected transferrin in rat liver

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.