Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies.

1. In the process of iron uptake by precursors of the erythrocytes probably more than one membrane component is involved; besides the specific transferrin receptor, another membrane component with a high iron activity after incubation with 59Fe can be isolated. 2. A striking resemblance exists between rat and human reticulocyte components which are involved in the process of iron uptake. 3. Incubation of reticulocytes with Fab fragments of an antibody against the membrane receptor for transferrin causes a concentration-dependent decrease in transferrin binding and iron uptake. 4. The membrane receptor complex isolated is still heterogeneous; analytical ultracentrifugation studies suggest a molecular weight lower than 230,000. 5. Intact immature red cells are necessary for specific binding of transferrin with the receptor followed by iron uptake. This is the only mechanism for iron uptake. 6. Immunofluorescence studies showed that the receptor for transferrin is localized at the outside of the cell membrane.

The uptake of iron by reticulocytes. The influence of purification of the ghosts on iron-containing components in the ghost suspension.

1. Rat reticulocytes previously incubated with ((59)Fe, (125)I)-labelled transferrin were hemolysed to yield labelled ghosts. 2. The solubilized ghosts can be fractionated, by gel filtration on Sepharose 2B and 6B, into several (59)Fe- and (125)I-containing compounds, classified as A, B(1) and B(2). 3. These fractions were prepared from ghosts which were obtained at different centrifugation rates and further purified by sucrose density gradient centrifugation in order to obtain membrane compounds purified from mitochondrial and lysosomal impurities. The influence of these purification steps on the appearance and the (59)Fe/(125)I activity of the three components was investigated. 4. The first Sepharose 2B fraction with high molecular mass, greater than 10(6) daltons, is an intracellular product of mitochondrial and lysosomal origin which precipitates with the membrane fractions during the preparation of the ghost. The first Sepharose 6B fraction, B(1), with M(r) approximately 10(6) seems to be a real membrane component. The second Sepharose 6B fraction, B(2), with M(r) approximately 230 000 represents a real membrane receptor transferrin.

The involvement of iron and lipid peroxidation in the pathogenesis of HCB induced porphyria.

Hexachlorobenzene (HCB) induces a porphyria characterized by a diminished activity of the enzyme uroporphyrinogen decarboxylase (URO-D), presumably due to inactivation by reactive metabolites of HCB. We studied the effect of iron on HCB porphyria in female rats, to determine whether the iron dependent process of lipid peroxidation was involved in the pathogenesis of porphyria. We showed that malondialdehyde formation is increased in rat liver tissue of porphyric rats and that high molecular weight proteins due to cross-linking are formed. We also showed that the induction of porphyria by HCB is dependent on the presence of iron. Our findings suggest that lipid peroxidation is involved in the toxicity of HCB and that the aggravating effects of iron on HCB are mediated by lipid peroxidation.

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution.

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.

Binding of human isotransferrin variants to microvillous and basal membrane vesicles from human term placenta.

Transferrin (Tf)-dependent iron transfer from mother to fetus is mediated by Tf receptors (TfRs) which are present on both microvillous and basal membranes of human placental syncytiotrophoblast. We used microvillous and basal membrane vesicles, both isolated from the same human term placenta, to investigate the binding of [125I]-labelled diferric bi-bi antennary tetra-sialo Tf (bb Tf), bi-tri-antennary penta-sialo Tf (bt Tf) and tri-tri-antennary hexa-sialo Tf (tt Tf). To diminish the effect of endogenous Tf, membrane vesicles were washed before binding of [125I]-Tf. The number of TfRs on microvillous membranes was 6.1 +/- 2.4 (mean +/- s.d., n = 15) times higher than that on basal membranes, whereas the affinity of TfRs on basal membranes was 3.9 +/- 0.4 (mean +/- s.d., n = 15) times higher than that of TfRs on microvillous membranes, irrespective the isoTf used. The affinity constants of TfRs on both microvillous and basal membranes were higher for bb Tf than for bt Tf and higher for bt Tf than for tt Tf. However, these latter differences were rather small and probably not of physiological importance.

Non-transferrin iron uptake by trophoblast cells in culture. Significance of a NADH-dependent ferrireductase.

This study shows that trophoblast cells in culture are able to take up 59Fe from both Fe(III)nitrilotriacetate (NTA) and Fe-ascorbate. Fe in the presence of ascorbate is assumed to be Fe(III) in equilibrium with Fe(II). Kinetic parameters for non-transferrin iron uptake are determined from initial rate experiments, yielding Vmax=366 pmol/mg protein/5 min and Km=0.96 microM for Fe(III)NTA and Vmax=4043 pmol/mg protein/5 min and Km= 1.3 microM for Fe-ascorbate. Since trophoblast cells in culture reduce extracellular Fe(III)CN, and uptake of 59Fe from Fe-ascorbate is higher than that from Fe(III)NTA, it is suggested that reduction of Fe(III) precedes uptake. Uptake of 59Fe from both Fe-ascorbate and Fe(III)NTA is inhibited by Fe(II)chelator ferrozine and membrane-impermeable Fe(III)CN, further supporting this hypothesis. Studies with microvillous membrane vesicles (MMV) and basal membrane vesicles (BMV) reveal the presence of a NADH-dependent ferrireductase. Reduction of Fe(III)CN follows Michaelis-Menten kinetics, both with respect to [NADH] and [Fe]. NADPH is ineffective as electron donor. The rate of Fe(III)CN reduction by BMV is 2.5 times higher compared to MMV, while Km values for Fe(III)CN and NADH are not significantly different. These results reveal that a transmembrane NADH-dependent ferrireductase plays a role in uptake of non-transferrin iron. The possibility that this enzyme system is involved in iron transfer across the basal membrane is discussed.

Serum ferritin and iron stores during pregnancy.

The iron status of two groups of pregnant women was investigated. One group did not receive iron (group B), the other erceived 100 mg iron/day (group A). 1. In all individuals concentrations of hemoglobin, serum iron, transferrin and serum ferritin were determined at regular intervals from the third month until delivery and at 3 months after delivery. The same determinations were performed in cord blood. 2. Changes in iron status appeared to be less in individuals with iron supplement than in those without iron supplement. A fall in Hb, serum iron and serum ferritin is observed in all individuals. 3. Three months after delivery the Hb concentration has generally returned to the normal female value, but the serum ferritin concentration is still very low. 4. The fetus does not discriminate as to the iron status of the mother. In both groups (A and B) cord blood values appeard to be not significantly different.

Regulation of transferrin receptor expression and distribution in in vitro cultured human cytotrophoblasts.

During gestation the transplacental iron transport is very important to the fetus. Iron uptake by the placenta can be studied in cultured cytotrophoblasts. The influence of culture time and human differic transferrin on the number and distribution of transferrin receptors (TfRs) was investigated in human cytotrophoblasts. Cytotrophoblasts cultured for 2.5 h had few TfRs (0.28 pmol/mg protein). With time, total TfR amounts increase (4.14 pmol/mg protein at 70 h). They increase to a higher level in cells cultured in iron-poor medium, indicating that iron has an effect on the TfR synthesis/breakdown ratio. TfRs were distributed between two 'active' (located at the cell surface and intracellularly) and one 'inactive' (located intracellularly) receptor pools. TfR distribution among these pools was modulated by culture time and iron. Trophoblasts regulated iron uptake by variation of number of surface TfRs via changes in total TfRs and redistribution of TfRs among the receptor pools.

Isotransferrins and pregnancy: a study in the guinea pig.

During pregnancy the serum isotransferrin pattern changes towards transferrins with more complex carbohydrate chains. The main pregnancy-related isotransferrin (TfFast) and the most common isotransferrin in the non-pregnant guinea pig (TfSlow) were isolated and characterized. TfSlow had one biantennary- and TfFast one triantennary glycan chain. Is there a functional explanation for this pregnancy-related shift towards more complex glycan chain structure? We studied this question in the context of maternal and fetal erythropoiesis. In vitro incubations of maternal bone marrow cells (MBMC) and fetal erythroid liver cells (FELC) with doubly labelled TfSlow and TfFast revealed only slight differences in affinities for the transferrin receptor. Ka(TfSlow) = 0.17 mumol/l; Ka(TfFast) = 0.15 mumol/l. MBMC and FELC had equal Vmax values both for TfSlow and TfFast. Vmax = 100 Fe atoms/transferrin receptor.hour. Irrespective the cell population TfSlow and TfFast showed equal rates for endo- and exocytosis. kendo. = 0.3750 min-1, kexo. = 0.1450 min-1. It is concluded that the described shift in isotransferrin pattern is not functionally related to maternal or fetal erythropoiesis.

Effects of iron supplementation on iron uptake by differentiating cytotrophoblasts.

Cultured in Medium-199, cytotrophoblasts isolated from human placentae differentiate morphologically (e.g. syncytium formation) as well as biochemically (e.g. expression of transferrin receptors, TfRs) into syncytiotrophoblast-like structures. The highest TfR numbers are observed in cells cultured in iron-poor culture medium. Investigated were the implications of the variation in surface TfR numbers on the uptake of iron by cytotrophoblasts cultured in iron-poor Medium-199. Despite differences in TfR densities induced by culture time and iron availability, the initial rate of iron uptake did not change (80-100 pmol/mg protein/h). Homeostasis of iron uptake could be explained by adaptive changes in the rate constant for TfR endocytosis (kend), exocytosis (kexo) and TfR cycle times. In undifferentiated cells (cultured for 18 h) kend was 0.299 min-1. In differentiated cells (culture time 65 h, higher surface TfR densities), kend changed to 0.138 min-1. Culture for 65 h in diferric transferrin-enriched medium resulted in intermediate TfR densities together with an intermediate kend (0.210 min-1). Adaptive changes in the corresponding rate constant of exocytosis were less pronounced (0.192, 0.192 and 0.260 min-1 respectively). It is concluded that differentiating cytotrophoblasts regulate iron uptake by variation of both TfR numbers and the rate of receptor-mediated endocytosis and exocytosis.

Morphological differentiation of cytotrophoblasts cultured in Medium 199 and in keratinocyte growth medium.

In culture, cytotrophoblast cells differentiate biochemically as well as morphologically into syncytiotrophoblast-like structures. Morphological and biochemical differentiation can be affected by the composition of the culture medium. The aim of this study was to analyze the morphological differentiation (syncytium formation) of cytotrophoblasts cultured in Medium 199 (M199) and keratinocyte growth medium (KGM). Term human cytotrophoblast cells were cultured in either M199 or KGM with daily refreshment of the media. Both media induced biochemical differentiation, as monitored by measuring hCG secretion. Syncytium formation was visualized by immunocytochemical staining of desmosomes (cell membranes). Cytotrophoblasts rapidly formed aggregates; however, single cells were seen throughout culture. Though the aggregates developed into syncytia, approximately 15% of the nuclei were still found in cell aggregates at the end of the culture period (4 days). The final percentage of nuclei in syncytia (60-70%) did not differ between the culture media used. Syncytium formation occurred more rapidly in KGM medium. Approximately 50% of the nuclei were found in syncytia after 40 and 50 h in KGM and M199, respectively. The number of nuclei per syncytium was slightly higher in M199, but the average surface area of the syncytia was larger in KGM cultured cells (162-132 mm2). These differences did not reach significance. We conclude that there is no major difference in morphological differentiation between cytotrophoblast cells in KGM or M199. Moreover, both media sustain equal rates of hCG secretion.

Observations on the iron status during pregnancy in rats. Iron transport from mother to fetus.

The objective of the present study was to investigate a transfer of liver iron in rats from mother to fetus during pregnancy. A decrease in maternal liver iron and an increase in fetal liver iron, in groups of 10 rats, have been shown, based on iron analysis and specific activity of 59Fe.

Regulation of transferrin receptor synthesis by human cytotrophoblast cells in culture.

The aim of this study was to examine the capacity of the syncytiotrophoblast to regulate transferrin receptor (TfR) synthesis in response to modulations in maternal iron supply. The model used was the primary trophoblast cell culture. Trophoblast cells isolated from term human placentas were cultured in iron-poor (Medium 199), iron-depleted (desferrioxamine (DFO)) and iron-supplemented (diferric transferrin (hTf-2Fe), ferric ammonium citrate (FAC) medium. TfR synthesis was reduced in response to hTf-2Fe supplementation. FAC did not modulate TfR synthesis. Iron deprivation by DFO resulted in clear stimulation of TfR synthesis. These results show that the differentiating trophoblast cells respond to pertubations in the (transferrin-mediated) iron supply by adjustments in the rate of TfR synthesis. Taking syncytiotrophoblast in culture as model for the maternal/fetal interface in vivo, our results would suggest that the placenta is able to make short term adjustments of the capacity for iron uptake.

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas.

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.

The effect of different iron compounds on transferrin receptor expression in term human cytotrophoblast cells.

During pregnancy, the mother is faced with an increased food demand. A good example of this increased demand is iron (Fe). Fe is needed in all growing cells. During pregnancy, the Fe transport to the fetus increases enormously. This amount can easily induce an Fe deficiency in the mother. Fe supplementation is very important for her, but not for the Fe status of the fetus, which is protected against Fe toxicity as well as deficiency. The placenta seems to be autonomous in Fe uptake. Likely there is a regulation mechanism. The human placenta is hemomonochorial. The cell layer of the fetus in contact with the maternal blood is formed by syncytiotrophoblasts. Fe is transported to the placenta by transferrin. Transferrin binds to a transferrin receptor on the trophoblast membrane and is internalized via an endocytic pathway. During this cycle, Fe is released from transferrin and the transferrin-transferrin receptor complex is recycled to the membrane. Isolated trophoblast cells from term placentas form a syncytium in vitro, and transferrin receptors are expressed. Expression depends on the number of cells in culture, culture time, the amount of Fe available, and the Fe compound. By regulation of the number of transferrin receptors, trophoblasts are able to control their Fe uptake.

Transport of transferrin-bound iron into rat Sertoli cells and spermatids.

Transferrin (Tf), a major secretory protein of Sertoli cells, may transport iron to spermatogenic cells. This was assessed by measuring the uptake of Fe from 59Fe-125I-labelled rat Tf by Sertoli cells and round spermatids in vitro. Uptake of Fe from labelled Tf by Sertoli cells after a 72-h pre-incubation period was linear for 20 h (approximately 18 pmol/10(6) cells/20 h), whereas the uptake of Fe from labelled Tf by round spermatids after a 16-h pre-incubation period reached a plateau by 2 h (approximately 5 pmol/10(6) cells/2 h). The corresponding net uptake of Tf by both cell types was less than 0.1 pmol. High speed supernatants prepared from Sertoli cells and spermatids labelled with 59Fe-125I-Tf were fractionated by gel permeation chromatography. Separate peaks of protein-bound 59Fe and 125I-Tf were observed. Protein bound 59Fe could be precipitated with an antiserum to rat ferritin. It is concluded that iron from exogenous Tf is transported into Sertoli cells and round spermatids in vitro, and is complexed to intracellular ferritin. However, the present results do not exclude the possibility that Sertoli cell Tf may serve purposes other than iron transport.

The iron status in healthy individuals aging from 18-25 years.

The haemoglobin, haematocrit, erythrocyte count, transferrin and serum iron values of a group of 65 healthy young people aging from 18-25 years were determined. Ferritin in serum was quantitated by radioimmunoassay to determine the usefulness of this assay in reflecting iron stores of healthy people.

Are lysosomes directly involved in the iron uptake by reticulocytes?

The mechanism by which iron is transferred from the plasma protein transferrin into erythroid precursors for incorporation in heme is not completely understood. To show a direct functional role of lysosomes in the process of iron uptake we tried to isolate lysosomes from reticulocytes, which have been incubated with 125ITf59Fe. However, with various cell fractionating techniques described for liver cells no pure lysosomes from reticulocytes could be obtained. Fluorescence and electron microscopy showed that reticulocytes hardly contain well-defined lysosomes. There are several indications that in reticulocytes acid vacuoles instead of lysosomes are involved in the removal of iron from endocytosed transferrin. The presence of apoTf, monoferric TfFe(A), monoferric TfFe(B) in the medium after incubation of reticulocytes with diferric transferrin, together with the fact that both iron binding sites of transferrin release their iron at pH present in acid vacuoles, suggests a second mechanism of iron uptake by reticulocytes, in which acid vacuoles are not involved.

Impaired iron uptake and transferrin binding by erythroblasts in the anaemia of rheumatoid arthritis.

Serum and bone marrow from 18 patients with rheumatoid arthritis (RA) and five healthy controls were studied in order to establish a possible role of impaired iron uptake and transferrin binding by erythroblasts in the pathophysiology of anaemia of chronic disease (ACD) in RA. Iron incorporation into erythroblasts was reduced in patients with ACD using a method based on incubation of erythroblasts with radiolabelled 59Fe-125I-transferrin. It correlated negatively with C-reactive protein (CRP). In iron deficient RA patients it tended to be reduced as well. These patients had the same level of RA disease activity as in ACD. Transferrin binding by erythroblasts was significantly impaired in ACD compared to controls, although it tended to be reduced in all RA groups. These findings suggest that impaired iron uptake by erythroblasts, probably due to decreased transferrin binding to erythroblasts, might be a pathophysiological factor in ACD in RA.