Ferritin in the red cells of normal subjects and patients with iron deficiency and iron overload

S ummary . Red cell ferritin was measured in normal subjects and patients with iron deficiency and iron overload by means of radioimmunoassays with antibodies to liver (basic) and heart (acidic) ferritins. In most of the subjects examined, red cells were found to contain greater amounts of heart-type than liver-type ferritin. The basic ferritin content reflected the abnormal body iron status both in iron deficiency and iron overload while the acidic ferritin content was less closely related to the iron status. The two immunologically different red-cell ferritins probably represent distinct ferritin molecules and may have different metabolic functions within haem-synthesizing erythroid cells.     

Red cell ferritin and iron overload in heterozygous beta-thalassemia

Red cell ferritin was evaluated in 101 individuals with heterozygous beta-thalassemia to determine its clinical utility as an index for iron deficiency or overload in these subjects. The mean red cell ferritin for the total population was elevated threefold and showed a significant correlation with transferrin saturation, plasma ferritin, and HbA2 levels. Five of six subjects with reduced red cell ferritin had associated iron deficiency; a further five had iron deficiency and normal red cell ferritin. Normal red cell ferritin occurred in 51 subjects, and 44 had increased values. In the elevated red cell ferritin group, 21 individuals had associated normal plasma ferritin, and 23 had increased plasma ferritin. Only in the latter group was red cell ferritin significantly correlated with transferrin saturation and plasma ferritin. Ten individuals had a red cell ferritin greater than or equal to 150 attogram/cell, and liver biopsy performed in four showed grades II to IV iron overload. A clinical feature of subjects with both increased red cell and plasma ferritin levels was a high incidence of inappropriate iron administration. These findings suggest that red cell ferritin, particularly when combined with plasma ferritin, is a useful parameter for determining potential iron overload in individuals with heterozygous beta-thalassemia.     

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.     

Red cell basic ferritin content of patients with megaloblastic anaemia due to vitamin B12 or folate deficiency

The basic ferritin content of red cells was measured in patients with untreated megaloblastic anaemia. The red cell ferritin of 10 patients with anaemia and vitamin B12 deficiency (mean 579, range 68-2616 attogram (ag)/cell); and of 8 patients with folate deficiency (mean 792, range 141-2373 ag/cell) were significantly elevated (P less than 0.001) compared with normal subjects (mean 10.7, range 4-47 ag/cell) and showed a significant correlation with pre-treatment levels of plasma ferritin and less so with percent transferrin saturation. Following vitamin replacement elevated red cell ferritin levels decreased during the period of reticulocytosis and was normal in 9 patients evaluated after 6 months. The magnitude of increase in red cell basic ferritin levels observed in untreated megaloblastic anaemia is comparable to that of subjects with idiopathic haemochromatosis and suggests that interpretation of this index for iron overload should take into consideration concomitant body folate or vitamin B12 status.     

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.     

Regulation of hepatic transferrin, transferrin receptor and ferritin genes in human siderosis

Although many studies have examined the regulation of transferrin, transferrin receptor and ferritin subunit gene expression in experimental systems, no molecular biological data in humans have been documented to date. In this study we simultaneously analyzed the hepatic content of transferrin, transferrin receptor and heavy and light ferritin subunit messenger RNAs in tissue samples obtained from subjects with normal iron balance and patients with primary or secondary iron overload. Steady-state levels of transferrin messenger RNA were not depressed by iron overload. On the contrary, they were increased (p less than 0.001) in patients with severe hepatic siderosis (liver iron content greater than 200 mumol/gm dry wt) as compared with the control group. This indicates that, as already suggested by our previous data in experimental siderosis, iron maintains the ability to induce transferrin gene activity even when cellular iron content is significantly increased. Transferrin receptor gene expression was found to respond in the same manner to any cause of iron-tissue load, regardless of the cause. In fact, a lower signal for transferrin receptor messenger RNA was consistently detected in iron-overloaded patients vs. control subjects, particularly in patients with thalassemia major and idiopathic hemochromatosis (p less than 0.001). Ferritin light-subunit messenger RNA accumulation was significantly increased in those patients with severe siderosis (idiopathic hemochromatosis and thalassemia major = liver iron between 200 and 600 mumol/gm dry wt). The fact that no significant change in hepatic ferritin heavy-subunit gene expression was detected in iron-loaded patients confirms preferential production of light-subunit--enriched ferritins in long-term iron overload.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular expression and regulation of iron transport and storage proteins in genetic haemochromatosis

: Genetic haemochromatosis is a common iron overload disorder of unknown aetiology. To characterize the defect of iron metabolism responsible for this disease, this study localized and semiquantified the mRNA and protein expression of transferrin, transferrin receptor and ferritin in the liver and duodenum of patients with genetic haemochromatosis. Biopsies were obtained from iron-loaded non-cirrhotic patients with genetic haemochromatotic and control patients with normal iron stores. Additional duodenal biopsies were obtained from patients with iron deficiency. Immunohistochemical and in situ hybridization analysis for transferrin, transferrin receptor and ferritin was performed. Hepatic transferrin, transferrin receptor and ferritin protein expression was localized predominantly to hepatocytes and was increased in patients with genetic haemochromatosis when compared with normal controls. Interestingly, hepatic ferritin mRNA expression was not increased in these same patients. In the genetic haemochromatotic duodenum, ferritin mRNA and protein was localized mainly to crypt and villus epithelial cells and the level of expression was decreased compared with normal controls, but similar to iron deficiency. Duodenal transferrin receptor mRNA and protein levels colocalized to epithelial cells of the crypt and villus were similar to normal controls. Early in the course of genetic haemochromatosis and before the onset of hepatic fibrosis, transferrin receptor-mediated iron uptake by hepatocytes contributes to hepatic iron overload. Increased hepatic ferritin expression suggests this is the major iron storage protein. While persisting duodenal transferrin receptor expression may be a normal response to increased body iron stores in patients with genetic haemochromatosis, decreased duodenal ferritin levels suggest that duodenal mucosa is regulated as if the patient were iron deficient.     

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.     

Relative iron deficiency in hereditary spherocytosis

Seventy-three patients with hereditary spherocytosis (HS) (58 nonsplenectomized, 15 splenectomized) were studied to evaluate iron status and the adequacy of iron availability for erythropoiesis. Splenectomized patients, who had hemoglobin levels in the normal or upper normal range, had higher levels of serum iron, transferrin saturation, and serum ferritin than normal matched controls and normal zinc protoporphyrin (ZnPP) levels. On the contrary, nonsplenectomized patients presenting with mild to severe anemia had higher red cell ZnPP concentrations than both splenectomized subjects and matched normal controls. ZnPP in nonsplenectomized patients correlated inversely with Hb concentration, mean corpuscular volume (MCV), mean red cell hemoglobin concentration (MCHC), transferrin saturation, and serum iron, and directly with reticulocyte count. At multiple regression analysis only Hb concentration was a significant explanatory variable for high ZnPP. The authors conclude that a number of nonsplenectomized HS patients have relative iron deficiency primarily because of expansion of erythropoiesis caused by anemia.     

Clinical significance of erythrocyte ferritin

Quantitative and qualitative evaluations of erythrocyte ferritin in 161 patients with RA and RAEB in MDS, AML, CML, PV, PA, HS, IDA, chronic liver disease and alcoholic liver disease were carried out. Mean erythrocyte ferritin levels of patients with RA, AML, PA, HS and alcoholic liver disease were increased compared with normal subjects. On isoelectric focusing analyses (IEF), erythrocyte ferritin in normal subjects were detected between pI 5.1 and 5.7. In the cases of RA, pI ranges of erythrocyte ferritin may be divided into three groups, acidic, neutral, basic shift on IEF respectively. In these groups, the more acidic the ferritin shift, the higher the proportion of morphological abnormalities of the erythroid precursors in the bone marrow was observed. In patients with AML (M2, M3, M4), little difference was found among these three subtypes, and all of the cases showed similar pattern with normal subjects on IEF. The ferritin from IDA showed low levels and slight basic shift compared with normal subjects on IEF, and these features were also found in patients with CML (chronic phase) and PV. After iron supplementation, marked increase of acidic ferritin was detected on IEF indicating an intermediate store for iron destined for haem synthesis. It was clear that the stainable iron in liver parenchymal cells were found at erythrocyte ferritin concentration 20 ag/cell or over in patients with chronic liver disease. Measurement of erythrocyte ferritin concentration is a helpful method for evaluating iron deposition in hepatocyte non-invasively. From these results it is considered that quantitative and qualitative analyses of erythrocyte ferritin are very useful for evaluating erythropoiesis as well as iron metabolism.     

Iron metabolism in the hemoglobin-deficit mouse: correlation of diferric transferrin with hepcidin expression

The iron requirements of the erythroid compartment modulate the expression of hepcidin in the liver, which in turn alters intestinal iron absorption and iron release from the reticuloendothelial system. We have taken advantage of an inherited anemia of the mouse (hemoglobin deficit, or hbd) to gain insights into the factors regulating hepcidin expression. hbd mice showed a significant anemia but, surprisingly, their iron absorption was not increased as it was in wild-type animals made anemic to a similar degree by dietary iron depletion. In wild-type mice hepatic hepcidin levels were decreased but in hbd animals a significant and unexpected increase was observed. The level of absorption was appropriate for the expression of hepcidin in each case, but in hbd mice did not reflect the degree of anemia. However, this apparent inappropriate regulation of hepcidin correlated with increased transferrin saturation and levels of diferric transferrin in the plasma, which in turn resulted from the reduced capacity of hbd animals to effectively use transferrin-bound iron. These data strengthen the proposal that diferric transferrin is a key indicator of body iron requirements.     

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.     

Use of a monoclonal antibody against human heart ferritin for evaluating acidic ferritin concentration in human serum

Immunoassays for acidic ferritins rich in H subunits have shown that these isoferritins are predominant in some cells such as monocytes and red blood cells but have provided conflicting results about their presence in human serum. We have used an immunoradiometric assay based on a monoclonal antibody against human heart ferritin (monoclonal 2A4) for evaluating acidic ferritin concentration in human serum. This assay proved to be highly specific for acidic isoferritins having more than 60% H subunits. Heart-type ferritin was detected in only one fifth of normal sera and sera from patients with iron overload; values were very low compared with those for basic ferritin. Acidic ferritin was found in relatively high concentrations in most patients with iron deficiency anaemia. In other disease states characterized by increased serum concentrations of basic ferritin, acidic ferritin was always less than 21% of the total ferritin. Dialysis in low-ionic-strength buffer showed that both normal and pathological sera had binding factors for human heart ferritin. We conclude that: (i) human serum contains low concentrations of acidic isoferritins which, at variance with basic ferritin, do not appear to be directly related to the amount of storage iron; (ii) the findings of the present study reinforce the opinion that basic and acidic ferritins have different functional behaviours.     

Anemia and impaired stress-induced erythropoiesis in aceruloplasminemic mice

Ceruloplasmin (Cp) is an abundant, copper-containing plasma protein with an important role in iron homeostasis. Patients with hereditary Cp deficiency have iron deposits in liver and other organs, consistent with impaired iron flux. The mild anemia reported in some patients suggests a possible role for Cp in iron delivery to red cell precursors during erythropoiesis. To investigate this function of Cp, we determined the hematologic parameters in Cp-deficient mice under normal conditions and after erythropoiesis-inducing stress. Cp(-/-) mice have below normal hematocrit, red cell hemoglobin and volume, and serum iron. Red cell number and turnover and reticulocyte counts were identical in Cp(-/-) and Cp(+/+) mice. Thus, Cp(-/-) have mild microcytic, hypochromic anemia consistent with normal red cell formation but defective iron availability. Cp(-/-) and Cp(+/+) mice subjected to phenylhydrazine-induced hemolytic anemia exhibited identical decreases in hematologic parameters, but Cp(-/-) mice showed diminished recovery after removal of the stress. Administration of purified human Cp or iron-saturated transferrin to Cp(-/-) mice partially restored hemoglobin formation in reticulocytes. The mild anemia in Cp(-/-) mice and the diminished response to stress may reflect inefficient recycling of iron between the reticuloendothelial and erythropoietic systems. Our findings suggest a role for Cp in erythropoiesis by providing sufficient iron to the erythroid tissue and that the requirement for Cp is raised after erythropoietic stress.     

Differential response of non-transferrin bound iron uptake in rat liver cells on long-term and short-term treatment with iron.

BACKGROUND: Uptake of non-transferrin-bound iron by the liver is important as a clearance mechanism in iron overload. In contrast to physiological uptake via receptor-mediated endocytosis of transferrin, no regulatory mechanisms for this process are known. This study compares the influence of long-term and short-term depletion and loading of hepatocytes with iron on the uptake of non-transferrin bound iron, its affinity, specificity and the interaction with the transferrin-mediated pathways. METHODS: Rats were fed iron-deficient, normal and 3,5,5-trimethylhexanoyl-ferrocene-containing diets to obtain livers with the corresponding desired status and the hepatocytes from these livers were used for transport studies. Hepatocytes from normal rats were depleted or loaded with iron by short-term treatment with desferrioxamine or ferric ammonium citrate, respectively. Uptake of non-transferrin bound iron was assayed from ferric citrate and from ferric diethylene triammine pentaacetate. RESULTS: Uptake of non-transferrin-bound iron in hepatocytes could be seen as consisting of a high-affinity (Km=600 nM) and a low-affinity component. Whereas in normal and in iron-starved rats the high-affinity component was more prominent, it disappeared altogether in hepatocytes from rats with iron overload resulting from prolonged feeding with TMH-ferrocene-enriched diet. Overloading also led to loss of inhibition by diferric transferrin, which occured in starved as well as normal cells. In contrast, short-term iron-depletion of isolated hepatocytes with desferrioxamine had only a weak stimulatory effect, whereas treatment with ferric ammonium citrate strongly increased the uptake rates. However, the inhibition by diferric transferrin also disappeared. In both cases, uptake of non-transferrin bound iron was inhibited by apotransferrin. CONCLUSIONS: Non-transferrin bound iron uptake in liver cells is apparently regulated by the iron status of the liver. The mode of response to iron loading depends on the method of loading in terms of time course and the form of iron used. It cannot be explained by the behavior of the iron regulatory protein, and it is complex, seeming to involve more than one transport system.     

Iron metabolism in the Belgrade rat

Iron metabolism in the Belgrade rat was examined in the intact animal and in the reticulocyte suspensions. The plasma iron turnover was increased. However, when allowance was made for the effect of the elevated plasma iron concentration, erythroid marrow capacity for iron uptake was at basal levels. Numbers of erythroid cells in marrow and spleen measured by the radioiron dilution technique were increased. Thus iron uptake was not proportionate to the erythroid hyperplasia in the b/b rat, despite a more than adequate plasma iron supply. This relative deficiency in iron uptake was reflected in a severe microcytosis and elevated red cell protoporphyrin. Reticulocyte incubation studies demonstrated an unimpaired uptake of the transferrin- iron-receptor complex but a marked reduction in iron accumulation. The diferric transferrin molecule, when it did give up iron within the cell, released both of its iron atoms so that only apotransferrin was returned to the media. In contrast to the nearly complete release of iron within the normal reticulocyte, the major portion of iron taken up by the Belgrade reticulocyte was returned to the plasma. The release mechanism that can be impaired in iron-deficient reticulocytes by EDTA or cadmium was shown to be affected by lower concentrations of these substances in the Belgrade reticulocyte. It is concluded that the Belgrade rat has an abnormality of iron release within the absorptive vacuole that is responsible for a state of intracellular iron deficiency, involving the erythron and other body tissues.     

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.     

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.