The Regulation of Iron Release from the Perfused Rat Liver

Summary Factors affecting iron efflux from the isolated perfused rat liver were studied following the intravenous administration of transferrin-⁵⁹Fe or transferrin-⁵⁵Fc administered to the rat from 1·5 h to 3·5 d before perfusion of the liver. The liver was perfused with rat red cells suspended either in rat plasma or Eagle's Basal Medium (EBM). The mean rate of efflux into a plasma pool containing normal iron and transferrin concentrations was 0·9% of the initial hepatic radioactive iron pool per hour. In EBM the average rate of efflux was 0·1%/h and this could be increased to the rate observed with plasma by the addition of apotransferrin. The rate of iron release from the liver in the presence of apotransferrin or other chelators was inversely proportional to the time of prelabelling. Maximal release rates were observed in livers perfused within 5 h of administering transferrin-⁵⁹Fe to the rat. The effect of apotransferrin on efflux into EBM was concentration dependent. However, the maximum release of liver iron by apotransferrin occurred at physiological apotransferrin concentrations and addition of apotransferrin to plasma produced no increase in the rate of iron efflux. The stimulation of iron release in EBM caused by apotransferrin could be reversed by reducing the unsaturated iron binding capacity of the perfusate, either by addition of iron or removal of apotransferrin. However, increasing the iron concentration in the perfusate by the addition of iron-saturated transferrin without any reduction in the unsaturated iron binding capacity additionally increased iron release into plasma and EBM. This presumably reflects the exchange of plasma transferrin-³⁶Fe for liver ⁵⁹Fe. Hence iron release measured in these studies represents the sum of two processes—net release of ⁵⁹Fe induced by apotransferrin and iron exchange between plasma and liver iron pools. Apotransferrin and desferrioxamine were equally effective, per unit iron binding capacity, in mobilizing liver iron, and may compete for the same parenchymal iron pool. This suggests that mobilization of iron by apotransferrin may depend solely onits ability to chelate ferric iron and not on a more specific ferroxidase activity or interaction with membrane receptors.     

Importance of anemia and transferrin levels in the regulation of intestinal iron absorption in hypotransferrinemic mice.

The hypotransferrinemic mouse (trf (hpx)) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass (59)Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf (hpx) animals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf (hpx) mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.     

Molecular aspects of the binding of absorbed iron to transferrin

S ummary . To study the molecular aspects of the binding of absorbed iron to plasma transferrin, ⁵⁹Fe with high specific activity was administered via intragastric tube to iron-deficient rabbits. The distribution of the absorbed ⁵⁹Fe among the molecular forms of iron-transferrin was analysed using urea-polyacrylamide gel electrophoresis. Absorbed iron was bound to circulating transferrin one atom at a time. In four out of five animals, absorbed iron was predominantly bound to the site in the N-terminal domain of the protein. Thus, the two sites of transferrin may differ in their ability to load absorbed iron.     

Iron uptake from plasma transferrin by the duodenum is impaired in the Hfe knockout mouse

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced iron absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a C282Y mutation in the HFE gene. The function of HFE protein is unknown, but it is hypothesized that it acts in association with beta(2)-microglobulin and transferrin receptor 1 to regulate iron uptake from plasma transferrin by the duodenum, the proposed mechanism by which body iron levels are sensed. The aim of this study was to test this hypothesis by comparing clearance of transferrin-bound iron in Hfe knockout (KO) mice with that observed in C57BL/6 control mice. The mice were fed either an iron-deficient, control, or iron-loaded diet for 6 weeks to alter body iron status. The mice then were injected i.v. with (59)Fe-transferrin, and blood samples were taken over 2 h to determine the plasma (59)Fe turnover. After 2 h, the mice were killed and the amount of radioactivity in the duodenum, liver, and kidney was measured. In both Hfe KO and C57BL/6 mice, plasma iron turnover and iron uptake from plasma transferrin by the duodenum, liver, and kidney correlated positively with plasma iron concentration. However, duodenal iron uptake from plasma transferrin was decreased in the Hfe KO mice compared with the control mice. Despite this difference in duodenal uptake, the Hfe KO mice showed no decrease in iron uptake by the liver and kidney or alteration in the plasma iron turnover when compared with C57BL/6 mice. These data support the hypothesis that HFE regulates duodenal uptake of transferrin-bound iron from plasma, and that this mechanism of sensing body iron status, as reflected in plasma iron levels, is impaired in HH.     

The significance of transferrin for intestinal iron absorption

A mechanism is proposed by which apotransferrin is secreted from mucosal cells, loaded with iron in the intestinal lumen, and then the intact complex is taken into the cell. Within the cell, iron is released and transferred to the blood stream, whereas iron-free transferrin returns to the brush border to be recycled. We have investigated this hypothesis by measuring intestinal absorption of radioiron and 125I-labeled plasma transferrin using tied-off gut segments in normal and iron-deficient rats. There was no absorption of diferric transferrin from the ileum, but high absorption from the duodenum and jejunum segments. Jejunal absorption occurred as a function of the dose offered and showed saturation kinetics. In normal animals, 4 micrograms of the 50 micrograms of transferrin iron was absorbed over 1 hr. In iron-deficient animals, mean values as high as 13 micrograms were observed. Radioiron content of the jejunal mucosa bore a linear relationship to the dose administered and was inversely proportional to the amount of iron entering the plasma. Recycling of transferrin was indicated by the presence of labeled apotransferrin in the lumen, first observed between 15 and 60 min after the injection of diferric transferrin. A high resistance of diferric and apotransferrin to proteolytic degradation within the gut lumen was demonstrated. Comparative studies with lactoferrin and ferritin disclosed poor availability of their iron for absorption. The small amount that was absorbed did not relate to the iron status of the recipient animal. These studies support the role of mucosal transferrin as a shuttle protein for iron absorption.     

Molecular ferrokinetics in the rabbit

S ummary . Using urea-polyacrylamide gel electrophoresis it has been possible to distinguish the molecular forms of transferrin in rabbit serum. When ⁵⁹Fe-labelled diferric transferrin is injected into normal, anaemic or hypertransfused, polycythaemic rabbits, iron is removed from diferric transferrin in essentially pairwise fashion. Exchange of iron between transferrin and tissues was also studied using predominantly monoferric transferrin labelled with ⁵⁹Fe or ¹²⁵I, and with ¹²⁵I-labelled apotransferrin. The return of iron from tissue stores to circulating transferrin occurs one atom at a time to either site of the protein and, possibly, in pairwise fashion as well. The rate of clearance of iron from diferric transferrin differs from that of monoferric transferrins, and the rates at which iron is returned to empty sites of transferrin also differ, so that serum iron is not a kinetically homogeneous pool in the rabbit.     

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/b2 globin gene deletion) mice

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb^th3/+ mice. Mice with the Hbb^th3/+ phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb^th3/+ mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.     

Uptake of transferrin by rat peritoneal macrophages

S ummary . Activated rat peritoneal macrophages bind ¹²⁵I-apotransferrin in a time and temperature-dependent process, the amount of transferrin taken up at 4°C amounting to only about 15% of that bound at physiological temperatures. Binding is reversible, saturable, and largely abolished by prior treatment of the cells with Pronase. A single class of high affinity binding sites is evidenced by Scatchard analysis, each cell binding about 110 000 apotransferrin molecules with an apparent affinity constant of 1.4 x 10⁶ 1 mol^-1. Macrophages are also capable of binding about one-third as much iron-saturated transferrin as iron-free transferrin. Since binding of neither form of the protein is influenced by the presence of the other, separate and independent binding sites for apotransferrin and iron transferrin are presumed to exist on the macrophage.     

Does plasma transferrin regulate iron absorption?

It has been suggested that transferrin that has recently donated its iron to receptor sites is 'activated' to take up iron more avidly from donor tissues. The hypothesis was tested in vitro in a system in which use was made of the different electrophoretic mobilities of normal and desialated transferrin. Recently desaturated transferrin and native apotransferrin were added in equal amounts to a solution of radioactive ferric citrate to produce various end saturations. The resultant mixture was electrophoresed on 5.4% polyacrilamide gel, which was then sliced and counted for 59Fe counts. The size of the 2 radioactive peaks was then compared and expressed as a ratio. Using this in vitro system no supporting evidence could be found for the hypothesis that diferric transferrin which has just donated its iron is able to bind available iron more avidly than native apotransferrin.     

The Role of Ascorbic Acid in the Metabolism of Storage Iron

S ummary . The role of ascorbic acid in the metabolism of storage iron was investigated in guinea-pigs. Ascorbic acid deprivation increased the total non-haem iron concentration in the spleen and reduced it in the liver, and in both organs ferritin was diminished and haemosiderin increased. Replacing the ascorbic acid restored the normal distribution of iron between the two storage compounds, and in the spleen the total storage iron concentration returned to control levels within 24 hr. Evidence was obtained in experiments with ⁵⁹Fe that the accumulation of iron in the spleen was due to a diminished release from reticulo-endothelial cells. When ⁵⁹Fe-labelled haemoglobin in denatured red cells was injected, release of the isotope was inhibited in scorbutic animals. In contrast, after injecting labelled transferrin, ⁵⁹Fe in the liver parenchymal cells was released to a greater extent than in normals. These observations may explain certain ferrokinetic peculiarities in patients with scurvy, and possibly also the predominantly reticulo-endothelial localization of the iron in Bantu subjects with siderosis.     

Transferrin receptor expression in rat liver: immunohistochemical and biochemical analysis of the effect of age and iron storage

Hepatic transferrin receptors were studied in normal male rats at 1 to 59 wk after weaning, using immunohistochemical and biochemical techniques. The number of transferrin receptors measured and the intensity of the staining in situ decreased rapidly during the first 10 wk of life and more slowly thereafter. Immunohistochemistry further demonstrated changes in the topographical and (sub)cellular localization of the transferrin receptor. In the young rat livers, staining was almost exclusively present on hepatocytes in acinar zone 2 + 3 in a honeycomb to sinusoidal pattern. With aging, a panacinar heterogeneous and mainly sinusoidal staining of hepatocytes was more frequent. Kupffer cell positivity was more obvious as compared with the young rat livers. The observed changes in transferrin receptor expression may partly be explained by age-dependent alterations in DNA synthesis and proliferative potential of the liver cells. A series of rats were iron loaded with carbonyl iron up to 39 wk and "unloaded" by administration of a normal diet during 20 wk. In these animals, serial histochemical studies showed predominantly parenchymal (7 to 14 wk), mixed parenchymal and reticuloendothelial (39 wk) and almost exclusive reticuloendothelial siderosis (59 wk). In the siderotic livers transferrin receptor numbers tended to be lower than in the controls with significant differences after 14 and 39 wk. Immunohistochemistry showed decreased parenchymal but increased reticuloendothelial transferrin receptor expression with iron load. After the period of unloading, parenchymal transferrin receptors were virtually absent despite the negligible siderosis of these cells. In contrast, siderotic reticuloendothelial cells were intensely positive. These findings support down-regulation of parenchymal transferrin receptor resulting from iron storage. However, the positivity of siderotic reticuloendothelial cells and the absence of re-emergence of parenchymal receptors in conditions of minimal parenchymal and prominent reticuloendothelial siderosis need further elucidation.     

Liposome-encapsulated Desferrioxamine in Experimental Iron Overload

S ummary . We have encapsulated desferrioxamine (DF) in multilamellar liposomes (ML) and unilamellar liposomes (UL). Liposomes were prepared either with or without a glycolipid, i.e. galactocerebroside (GC). The average diameter of ML was 0.5 μm, and that of UL was 0.08 μm. Less than 5% of DF leaked out from the liposomes after incubation in mouse plasma for 6 h. ⁵⁹Fe-ferritin and ⁵⁹Fe-labelled heat damaged erythrocytes (⁵⁹Fe-DRBC) were administered to normal and hyper- transfused mice as models of iron overload. Ferritin was used to label liver parenchymal cells and DRBC to label the Kupffer cells of the liver. A single injection of ML or UL with or without galactocerebroside into normal and hypertransfused mice enhanced from 3- to 15-fold the urinary excretion of radioiron from ⁵⁹Fe-ferritin and from ⁵⁹Fe-DRBC injected mice. For both the normal and hypertransfused mice, liposomes containing GC removed more ⁵⁹Fe radioactivity from ⁵⁹Fe-ferritin injected mice than liposomes without the glycolipid; whereas liposomes without GC removed more ⁵⁹Fe radioactivity from mice receiving ⁵⁹Fe-DRBC. Thus GC-liposomes may have a higher affinity for parenchymal cells of the liver, whereas liposomes without the glycolipid may have a higher uptake by the Kupffer cells.     

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.     

Uptake of Transferrin-Bound Iron by Rat Cells in Tissue Culture

The in vitro uptake of transferrin-bound iron by rat liver cells and rat embryo cells in culture, and by rat reticulocytes, was studied using labelled rat serum. The uptake of iron by rat liver cells was linear with time and showed a curvilinear relationship with the serum iron concentration. Over 40% of the cell radioactivity was found to be ferritin associated. Incubation of rat transferrin doubly labelled with ⁵⁹Fe and ¹²⁵l showed a difference between the uptakes of the two isotopes suggesting reflux of transferrin from the cell after iron uptake. In order to compare iron uptake from half saturated (Tf-Fe 1) and fully saturated (Tf-Fe2) transferrin, the three cell systems were each incubated in sera labelled with ⁵⁵Fe at 10% saturation and with ⁵⁹Fe at varying iron saturations ranging from 10% to 90%. Similar experiments were performed with sera having been randomly labelled with ⁵⁵Fe and labelled with ⁵⁹Fe but preincubated with reticulocytes in order to produce iron bound to non-reticulocyte orientated transferrin binding sites. The data obtained confirms the presence of a functional heterogeneity with respect to the half and fully saturated transferrin. Thus iron uptake from fully saturated transferrin was almost twice that for the half saturated molecule with rat liver cells or reticulocytes, and over three times that of the half saturated molecule with rat embryo cells. In contrast no such differences could be detected when iron uptake from serum randomly labelled with iron and serum with labelled non-reticulocyte orientated binding sites were compared. Finally, the effect of pre-incubating each of the three cell systems in unlabelled sera of varying percentage iron saturations on subsequent radio-iron uptake was studied. An inverse relationship between percentage saturation of the preincubation serum and subsequent uptake was demonstrated indicating that cellular mechanisms also influence cell iron uptake.     

Effect of repeated apotransferrin administrations on serum iron parameters in patients undergoing myeloablative conditioning and allogeneic stem cell transplantation

Myeloablative conditioning prior to allogeneic stem cell transplantation causes a rapid increase in transferrin saturation and potentially toxic non-transferrin-bound iron (NTBI) in plasma. We have studied the ability of repeatedly administered apotransferrin to maintain this iron in a transferrin-bound form. Twenty adult patients undergoing myeloablative conditioning and allogeneic stem cell transplantation were enrolled to receive apotransferrin with one of three dosage regimens. Ten consecutive patients with the same preconditioning were studied as controls. At the highest dose level, full transferrin saturation and appearance of NTBI were prevented in five of the eight patients. Serum iron increased significantly more in the patients receiving apotransferrin than in the controls and remained elevated until erythropoietic recovery. From the increment of iron saturation and the amount of endogenous and administered apotransferrin, an average 180 mumol of iron per day was bound to transferrin during the first 4 d after the start of the conditioning therapy. Thereafter, iron accumulation levelled off in most patients. The results suggested that about half of the amount of iron normally transported to erythropoiesis was initially released to plasma after induction of the erythroid arrest. Complete iron binding with apotransferrin would apparently require very high apotransferrin doses.     

Interaction of human diferric transferrin with reticulocytes.

Methods have been devised for preparing human transferrin with a different isotope of iron selectively labeling each of the two iron binding sites and for determining the distribution of radioiron among transferrin molecules. When diferric human transferrin was exposed to human or animal reticulocytes, there was an equal contribution of radioiron from the acid-stable and acid-labile sites. In this delivery, both atoms of iron were removed simultaneously from the diferric transferrin molecule, converting it to apotransferrin. At similar iron concentrations the amount of iron delivered by diferric transferrin was twice that delivered by monoferric transferrin.     

Transferrin uptake by bone marrow macrophages is independent of the degree of iron saturation

The uptake of transferrin by macrophages was studied in relation to the degree of iron saturation. Rat bone marrow derived macrophages were incubated with transferrin labelled with 59Fe and 3H. At 37 degrees C the amount of 59Fe incorporated by macrophages was dependent on the time of incubation. 3H labelled transferrin was found degraded in the supernatants of the cell culture (material not precipitated by trichloroacetic acid) in a time dependent fashion. Taking into account the specific activity of 59Fe-3H labelled transferrin, we found that 95% of the transferrin uptake was degraded. This suggests that most of the uptake of transferrin was not mediated by a receptor-dependent mechanism, but by a phase fluid endocytosis. 3H-labelled apotransferrin appears in the supernatant of the cell culture at the same rate as 59Fe-3H labelled diferric transferrin, showing an identical uptake for the two types of transferrin. Uptake of apo- or diferric transferrin by macrophages was identical in relation to time of incubation and the amount of transferrin used. These studies suggest that most of the transferrin uptake by bone marrow macrophages (non-activated or non-elicited cells) is mediated by a non-receptor mechanism that is independent of the degree of transferrin saturation.     

Erythrokinetics and ferrokinetics of a viral-induced murine erythroblastosis

A variant of Rauscher leukemia virus, designated RLV-A, induced a slow progressive impairment of erythropoiesis in BALB/c mice. Identified in this study were a shortened red cell 51-cr half-time, anemia with indices showing minimal but significant hypochromia, ineffective erythropoiesis, and infiltration of the liver, spleen, and peripheral blood with erythroid pecursors. Ferrokinetic studies indicated a normal plasma iron turnover in infected mice but a decreased red cell iron turnover. Large amounts of 59Fe were taken up by the enlarged liver and spleen. Peak splenic heme 59Fe synthesis was delayed 12 hr in the infected mice. The substantial increase in the splenic intraerythrocytic nonheme iron pool and the hypochromic indices indicate a process analogous to that seen in the sideroblastic anemias. The disease produced by this RLV-A variant may prove useful for studying various aspects of the preleukemic sideroblastic anemias and DiGuglielmo syndrome.     

Hepatic Iron Deposition in Humans: I. First-Pass Hepatic Deposition of Intestinally Absorbed Iron in Patients with Low Plasma Latent Iron-Binding Capacity

In patients with spontaneous chronic low plasma latent iron-bindingcapacity, a large fraction of the intestinally absorbed iron is deposited in theliver without prior appearance in the systemic plasma. Such hepatic iron isnot released to any large extent during the subsequent 2 weeks.

Our results are consistent with the hypothesis that iron absorbed from theintestinal tract is not initially bound to transferrin. When the plasma latentiron-binding capacity is normal, iron binds to transferrin prior to reachingthe liver; when the plasma latent iron-binding capacity is low, a portion ofthe iron reaches the liver either unbound or abnormally bound to transferrin,resulting in its immediate hepatic deposition.

Submitted on November 21, 1966 Accepted on March 19, 1967