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.     

Fluorescence probe measurement of the pH of the transferrin microenvironment during iron uptake by rat bone marrow erythroid cells

Fluorescence probe measurements of the transferrin micro-environment during iron uptake by rat erythroid cells revealed that part of the transferrin is taken up in an acidic environment. The pH of this intracellular transferrin environment is 5.7. When rat erythroid cell precursors are incubated with diferric transferrin then in the incubation medium monoferric transferrins TfNFe and TfFeC appear. In view of the known instability of TfNFe at acidic pH, TfNFe cannot arise after endocytosis of Tf2Fe in acid vesicles at pH below 6.0. The results support the existence of a mechanism other than endocytosis in the iron uptake process in rat erythroid cells.     

In vitro and in vivo studies of iron delivery by human monoferric transferrins

S ummary . According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. In this study we present the results of an evaluation of this hypothesis in vitro and in vivo with human pure monoferric transferrins obtained by preparative isoelectric focusing in granulated gels. The uptake of iron from monoferric transferrins TfFe_c and Fe_NTf by erythroid bone marrow cells, hepatocytes and stimulated T-lymphocytes in vitro was equal, even when both monoferric transferrins were present together in the incubation medium. Ferrokinetic studies in vivo , performed with both pure monoferric transferrins, showed that transferrin TfFe_c, as well as transferrin Fe_NTf, mainly deliver their iron to the erythron. As red cell ⁵⁹Fe utilization, red cell iron turnover and other ferrokinetic parameters, obtained from this study, were identical too it is evident that both iron-binding sites of transferrin are functionally homogeneous in vivo , with respect to iron delivery.     

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.     

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.     

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.     

Biologic and clinical significance of red cell ferritin

Red cell ferritin was measured in normal subjects and patients with disorders of iron metabolism, inflammation, liver dysfunction, impaired hemoglobin synthesis, and increased red cell turnover by means of radioimmunoassays with antibodies to liver (basic) and heart (acidic) ferritins. The normal mean values for basic and acidic ferritin were 8.9 and 22.7 altogram (ag)/cell, respectively. The red cell ferritin content reflected changes occurring in tissues both in iron deficiency and iron overload. Basic ferritin was more closely related to the body iron status than acidic ferritin, and the acidic/basic ferritin ratio was increased in iron deficiency and decreased in iron overload. The major factor determining the red cell ferritin content appeared to be the transferrin saturation, that is, the distribution of iron between monoferric and diferric transferrin. This is in keeping with recent data indicating a competitive advantage of diferric transferrin in delivering iron to erythroid cells. In addition, the red cell ferritin content was increased in thalassemic patients with normal iron status, appearing to be inversely related to the rate of hemoglobin synthesis. The determination of red cell ferritin, based on a commercially available basic ferritin assay, can have clinical application. It can be used for evaluating the adequacy of the iron supply to the erythroid marrow, particularly in patients with increased red cell turnover. Moreover, it may be useful in evaluating the body iron status in patients with hemochromatosis and liver disease.     

Quantitation of apo-, mono-, and diferric transferrin by polyacrylamide gradient gel electrophoresis in patients with disorders of iron metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Nonrandom distribution of iron in circulating human transferrin

By combining the urea gel electrophoresis technique of Makey and Seal with Western immunoblotting, a method has been developed for analyzing the distribution of iron between the two sites of circulating human transferrin. The new method avoids exposure of samples to a nonphysiologic pH that may promote removal or redistribution of iron from the protein; this facilitates examination of multiple samples at one time. Analysis of 21 freshly drawn specimens from normal human subjects confirms previous reports that iron is not randomly distributed in the specific sites of transferrin. Rather, there is a considerable range in the ratio of occupancies of N-terminal and C- terminal sites (N:C ratio), from 0.31 to 6.87 in the present study, with the N-terminal site predominantly occupied in most subjects. The N:C ratio correlates modestly with serum iron concentration (r = .54). Possible flaws in studies indicating a random occupancy of the specific sites of circulating transferrin may lie in the low pH to which samples may be exposed during procedures based on isoelectric focusing or in drawing inferences from data considering only total monoferric transferrin rather than the two distinguishable monoferric species.     

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.     

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.     

A reversible defect of platelet PDGF content in myeloproliferative disorders

The transport of iron through erythroid cell membrane was studied in a model system, measuring ferrous iron uptake by reticulocytes. It was found that these cells were able to take up ferrous iron and to incorporate it into haem at a rate similar to that observed when diferric transferrin was the iron donor. No comparable iron uptake could be measured when the metal was provided as Fe3+-citrate or when reticulocytes were replaced by mature erythrocytes. The involvement of endogenous transferrin in the Fe2+ uptake by reticulocytes could be excluded, since proteolytic treatment of the cells had no significant effect on the process. Fe2+ uptake by reticulocytes followed saturation kinetics, characteristic to carrier mediated transport processes. Kinetic analysis of the data revealed the following apparent transport parameters: Km = 8.8 +/- 3.8 microM; Vmax = 1.1 +/- 0.2 ng/10(8) reticulocytes/min. These results indicate that a high affinity, carrier mediated iron transport system is present in the reticulocyte membrane, ensuring the efficient translocation of the metal through the membrane barrier between the site of its release from transferrin and the site of its utilization.     

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.