Uptake and release of iron from human transferrin.

Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferic and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that of the acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 50Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin.     

The behavior of transferrin iron in the rat

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.     

Functional heterogeneity of transferrin-bound iron: iron uptake by cell suspensions from bone marrow and liver and by cell cultures of fibroblasts and lymphoblasts.

According to the hypothesis of Fletcher and Huehns, functional differences exist between both iron-binding sites of transferrin. The site designated A should mainly be involved in the delivery of iron to erythroid cells, whereas site B should donate its iron preferentially to cells involved in the absorption and storage of iron. In the present study this hypothesis could be confirmed by in vitro experiments with various cell types. Iron transferrin preincubated with rat bone marrow cells donates less iron to rat bone marrow cells, Chinese hamster fibroblasts, human fibroblasts and human lymphoblasts than freshly prepared iron transferrin equal in iron and transferrin concentraion. Rat liver parenchymal cells, however, take up more iron from preincubated than from freshly prepared iron transferrin. Obviously, site A not only donates iron preferentially to erythroid cells but also to (rapidly) dividing nonerythroid cells in culture. From experiments with iron transferrin mixtures in which radioiron was present at low or high iron saturation, it could be concluded that rat bone marrow cells take up iron equally well from monoferric as from diferric transferrin. The observed functional heterogeneity could, therefore, not be ascribed to differences between monoferric and diferric transferrin.     

The behavior of asialotransferrin-iron in the rat

The effect of desialylation of rat and human transferrins on hepatocyte processing of the protein and its iron was studied in rats. No alteration in early transferrin catabolism was observed. Radioiron disappearance from the plasma and liver iron uptake were more rapid for asialotransferrins than for normal transferrins (P less than .001). Furthermore, radioiron plasma clearance of human tri-sialotransferrin was faster (P less than .05) and liver uptake higher (P less than .002) than for human pentasialotransferrin. When the asialoglycoprotein receptor was blocked by the prior injection of asialofetuin, asialotransferrin behaved like normal transferrin. When the transferrin receptor was blocked by the prior injection of 50 mg human diferric transferrin, iron uptake from all transferrins was delayed to such an extent that uptake through both receptors seemed to be affected. Approximately 90% of the hepatic radioiron from all transferrins was chelated by desferrioxamine and excreted into the bile, indicating its uptake by the hepatocyte rather than the reticuloendothelial (RE) cell. The rate of iron release into the plasma and its subsequent accumulation in the red cell mass over a 2-week period was similar for human asialotransferrin, ferritin, and hemoglobin iron. This study 1) confirmed that asialotransferrin-iron uptake by the hepatocyte is mediated by both transferrin and asialoglycoprotein receptors; 2) demonstrated that not only asialotransferrin but also transferrin of low sialic acid content will increase iron turnover and lead to excessive iron loading of the hepatocyte; 3) and showed that the intrahepatocyte metabolism of asialotransferrin-iron did not differ from that of iron delivered by normal transferrin.     

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.     

Evidcnce for the Functional Heterogeneity of the Two Sites of Transferrin in Vitro

A recently developed crossed immunoelectrophoretic method for displaying and quantitating the four possible molecular species of transferrin has been utilized to assess the relative effectiveness of each site of rabbit and human diferric transferrin in providing iron to rabbit reticulocytes. The site which appears to reside in the N-terminal half of the rabbit protein was found to be at least 5 times more effective than its counterpart. However, both sites may serve as iron donors in monoferric as well as diferric rabbit transferrins. It is also possible that iron may be removed from rabbit transferrin in pairwise as well as sequential fashion. In human diferric transferrin, the site in the C-terminal domain functions as the better iron donor for rabbit reticulocytes.     

Competitive advantage of diferric transferrin in delivering iron to reticulocytes.

Radioiron- and radioiodine-labeled forms of human diferric and monoferric transferrin and apotransferrin, isolated by preparative isoelectric focusing, were used to define transferrin-iron uptake by human reticulocytes. In mixtures of human diferric and monoferric transferrin, the diferric molecule had a constant 7-fold advantage in delivering iron to reticulocytes, as compared with the 2-fold advantage when single solutions of mono- and diferric transferrins were compared. This was shown to be due to competitive interaction in iron delivery, probably at a common membrane-receptor binding site for transferrin. Apotransferrin did not interfere with the iron-donating process and its limited cellular uptake was inhibited in noncompetitive fashion by diferric transferrin.     

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.     

Distribution of iron between the binding sites of transferrin in serum: methods and results in normal human subjects.

When it is incompletely saturated with iron, transferrin may exist in four molecular forms: apotransferrin, monoferric (A) transferrin (with iron occupying only the A site of the protein), monoferric (B) transferrin, and diferric transferrin. By combining electrophoresis in urea-polyacrylamide gels with crossed immunoelectrophoresis using specific antihuman transferin antiserum, it is possible to display and estimate the concentration of each of these four forms in normal human serum. The distribution of iron between the binding sites of transferrin is neither random nor determined by the relative binding strengths of transferrin's two sites. Rather, the more weakly binding and acid-labile B site of the protein is predominantly occupied.     

Iron metabolism in normal and hemochromatotic macrophages

Certain metabolic pathways of iron were studied in macrophages (cultured human monocytes) obtained from normal and hemochromatotic subjects. The relative abilities of the hydrophobic ferrous chelator 2,2' bipyridine and the hydrophilic ferric chelators desferrioxamine (DFO) and diethylenetriaminepenta-acetic acid (DTPA) to release iron from normal and hemochromatotic macrophages which had previously been loaded with diferric transferrin were tested but there were no differences between the two groups. The relative affinity of the macrophages for diferric transferrin was next studied. Although the hemochromatic macrophages had a somewhat lower affinity for diferric transferrin iron than normal macrophages (Kd 4.7 x 10(-8) M vs. 3.0 x 10(-8)M) the difference did not reach statistical significance (t = 2.01013; P less than 0.07). In a further experiment there was no evidence that apotransferrin was directly involved in the release of iron from hemochromatotic macrophages. A clue to the nature of postendocytotic trans-membrane transport of iron was provided by the finding that it was inhibited by the hydrophobic ferrous chelator 2,2' bipyridine. However, the degree of inhibition was similar in both normal and hemochromatotic macrophages. In summary, none of the metabolic processes examined in the present study was abnormal in cultured human blood monocytes from hemochromatotic subjects.     

Occupancy of the iron binding sites of human transferrin.

The in vivo distribution of iron between the binding sites of transferrin was examined. Plasma was obtained from normal subjects under basal conditions and after in vitro and in vivo iron loading. Independent methods, including measurement of the transferrin profile after isoelectric focusing and cross immunoelectrophoresis, and determination of the iron content in the separated fractions were in agreement that there was a random distribution of iron on binding sites. This held true with in vitro loading, when iron was increased by intestinal absorption and with loading from the reticuloendothelial system. The data indicate that the distribution of apo-, monoferric, and diferric transferrins is predictable on the basis of the plasma transferrin saturation and negate the concept that iron loading of transferrin in vitro is a selective process with possible functional consequences in tissue iron delivery.     

Plasma radioiron kinetics in man: explanation for the effect of plasma iron concentration.

The plasma iron turnover was measured in 19 normal subjects. A correlation was found between plasma iron concentration and plasma iron turnover. In addition to the turnover of 55Fe at normal plasma iron concentration (predominantly monoferric transferrin), a second turnover in which the labeled plasma was saturated with iron (to produce predominantly diferric transferrin) was studied with 50Fe. It was demonstrated that diferric transferrin had a greater rate of iron turnover but that the distribution between erythroid and non-erythroid tissues was unchanged. It was concluded that plasma iron turnover is dependent on the monoferric/diferric transferrin ratio in the plasma but that the internal distribution of iron is unaffected.     

The use of reticulocytes with high non-haem iron pool for studies of regulation of haem synthesis

Summary One-hour incubation of reticulocytes with 10^?2m isonicotinic acid hydrazide (INH) and transferrin-bound ⁵⁹Fe changes the normal distribution of radioiron inside the cell. About 10% of ⁵⁹Fe is found in haem and 90% is present in the non-haem iron pool. The accumulated non-haem radioiron may be utilized for haem synthesis. This is demonstrated by the reincubation of washed reticulocytes with a high non-haem radioiron pool induced by INH under optimal conditions. The incorporation of radioiron from intracellular non-haem pool into haem is used as a method for the estimation of the rate of haem synthesis in the presence of various inhibitors. INH reduces haem synthesis from non-haem iron to a greater extent than that from transferrin iron. On the other hand, haemin, which inhibits the incorporation of ⁵⁹Fe from transferrin into haem, does not significantly decrease the utilization of intracellular non-haem iron for haem synthesis. These results are considered as further evidence for the inhibitory effect of haem on the membrane transport of iron. Cells with an artificially increased non-haem iron pool incorporate more [2-¹⁴C]glycine into haem than normal reticulocytes. These results are in accordance with the possibility that the supply of iron to the critical sites of haem synthesis may be a limiting factor controlling the rate of haem synthesis.     

Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.     

The Effect of pH upon Human Transferrin: Selective Labelling of the Two Iron-binding Sites

The influence of pH changes upon the iron-binding properties of transferrin was investigated in the absence of chelating agents. The effects were demonstrated by spectrophotometry, gel filtration, and by studies of the intermolecular transfer of 59Fe from transferrin to conalbumin. At pH values below 6.7, diferric transferrin readily loses iron. The monoferric molecule, which is relatively resistant to acid dissociation, is preferentially formed. A temporary reduction of pH provides a simple method for selectively attaching iron to one metal-binding site, and allows double isotopic labelling of the transferrin molecule. This technique may permit further investigation of the physiological properties of the two iron-binding sites.     

Failure of transferrin to enhance iron absorption in achlorhydric human subjects

There is evidence in experimental animals that transferrin, produced either by gastrointestinal cells or derived from bile, mediates the luminal absorption of iron. The applicability of these findings to human subjects was tested by administering diferric transferrin labelled with 3 mg 59Fe to seven patients with pernicious anaemia. Achlorhydric subjects were chosen to ensure that the iron transferrin complex did not dissociate in the stomach. The geometric mean absorption of 1.4% was similar to that of 3 mg iron given as ferric chloride (1.9%) and much less than that of ferrous ascorbate (18.9%). These findings suggest that transferrin does not play a physiological role in the absorption of iron in human subjects.     

Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.     

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.     

Mucosal iron transport by rat intestine

Using highly sensitive 2-site immunoradiometric assays, we examined the relationship between iron absorption from closed intestinal loops and transferrin and ferritin concentrations in isolated duodenal mucosal cells. As in prior studies, mucosal ferritin correlates inversely with iron absorption and directly with body iron stores as measured by the concentration of nonheme iron in liver. Mucosal transferrin, on the other hand, varies directly with both the total mucosal uptake of radioiron and the proportion of this radioiron transferred from the mucosa to the carcass. The highest correlation with iron absorption was observed with the transferrin-ferritin ratio in isolated mucosal cells. These results suggest that there are two functionally distinct iron- binding compartments in the duodenal mucosa. One is a strong compartment, ferritin, and the other is a transport compartment, transferrin. Control of iron absorption by the intestinal mucosa is closely tied to the balance between these two intracellular iron compartments.     

Translocation of different forms of transferrin from blood to bile in the rat

Five different forms of transferrin (rat apo [iron-free], rat diferric, diferric rat asialo, human diferric, and diferric human asialotransferrin type 3) were used to monitor the passage of this protein and its metal to the bile. Cumulative biliary excretion of the dose over 3 hours was determined. In addition, an excretion profile was constructed from the concentration of tracer in bile samples collected over 10-minute intervals. The profile obtained with apotransferrin was very similar to that found in an earlier study with albumin, the implication being that the apo form is transferred passively (e.g., by diffusion). Behavior of rat diferric transferrin, however, was consistent with the assumption that this form is transferred both passively and actively (i.e., in vesicles). The three other transferrins were investigated with the intent of broadening the spectrum of ligand affinities for the plasmalemma of hepatocyte. The higher this attraction was, the larger fraction of the dose appeared in bile. When transferrin was targeted to lysosomes, the bile contained several intermediate proteolytic fragments. Double-labeled (¹²⁵I, ⁵⁹Fe) transferrin was used to measure recovery of iron (Fe) relative to the protein (P) in bile. With rat diferric transferrin, the Fe/P ratio was 0.72. Lower values were recorded with transferrins (human or asialo) that had higher affinities for the plasmalemma and therefore were expected to be transported to a larger extent in vesicles. Of the biliary ⁵⁹Fe, 85% to 92% was protein bound. The proportion of the protein-bound fraction was essentially independent of the magnitude of Fe/P ratios.