Erythroblast Iron Metabolism in Sideroblastic and Sideropenic States

Iron appears to exert self-regulatory control over erythroblast iron uptake, iron storage and its incorporation into haem. It does this via iron regulatory proteins (IRPs) which bind reversibly to the iron responsive elements (IREs) on the mRNA of transferrin receptor (TfR), erythroid 5-aminolaevulinic acid synthase (ALA-S2) and ferritin. Iron deficiency leads to the binding of IRP to IRE. This binding inhibits the translation of mRNA for ALA-S2 and ferritin but stabilizes mRNA for TfR expression.

Sideroblastic erythropoiesis is highly ineffective and characterized by mitochondrial iron loading. The study of X-linked sideroblastic anaemia has shown that the entry of iron into the mitochondria is poorly controlled and able to occur when protoporphyrin production is reduced, as is seen with the ALA-S2 mutations, or when it is increased as has been seen with ABC7 transporter mutations.

Sideropenia characterises both iron deficiency anaemia (IDA) and the anaemia of chronic disease (ACD). Erythroblasts in ACD seem doubly equipped to protect their iron supply with their ability to increase the efficiency of transferrin-iron uptake as well as to activate the IRP/IRE system to increase surface TfR production. This increase in efficiency restricts the need to increase surface TfR production and maintains serum soluble TfR (sTfR) values within the normal range in iron replete ACD. The coexistence of iron deficiency with chronic disease, however, is associated with an increase in both the efficiency and number and a highly significant rise in sTfR values.     

Clinical importance of determining levels of circulating transferrin receptors in blood

Circulating serum transferrin receptor level was measured using mouse monoclonal antibody against transferrin receptor (Orion Diagnostica, Finland) in 126 patients with various disorders of erythropoiesis and the results were compared to those obtained form control group consisted of 30 healthy volunteers with normal iron stores. Serum transferrin receptor level was significantly elevated in patients with iron deficiency and in all patients with hyperplastic erythropoiesis (hereditary spherocytosis, immune hemolytic anemia, beta thalassemia, myelodysplasia). Measurement of circulating serum transferrin receptor level was a sensitive indicator of iron depletion as well as a helpful parameter in differential diagnosis between iron deficiency and anemia of chronic disease where circulating transferrin receptor level was not elevated. Index transferrin receptor/ferritin calculated as a ratio of circulating serum transferrin receptor level to log serum ferritin level was a more sensitive parameter than measurement of serum transferrin receptor not only for determination of patients with anemia of chronic disease, but also for discrimination of patients with elevated serum transferrin receptor level due to true iron deficiency from those with high serum transferrin receptor level caused by relative iron deficiency in hyperplastic erythropoiesis.     

Expression and extracellular release of transferrin receptors on erythropoiesis

One of the most important factors for the proliferation and hemoglobin synthesis of erythroid cells is iron atom. This atom is tightly bound to serum transferrin (Tf) and is taken up by erythroblasts and reticulocytes through transferrin receptor (TfR). Both Tf and TfR are reutilizable and have roles for the efficient intracellular accumulation of iron. In addition to the reutilization (recycling), the expression of TfR is also regulated by cytoplasmic iron concentration; the increase of iron downregulate the synthesis of TfR at the translational level and vice versa. This mechanism was recently explained by the binding between "iron responsive element (IRE)" in the 5' end of TfR mRNA and IRE binding protein by a transacting manner. Johnstone et al, and we found that TfR was externalized from sheep reticulocyte and human erythroleukemia cell, K562, respectively. Furthermore, we confirmed that this shed TfR was detected in blood and concluded that the quantitation of TfR in serum is a useful index for evaluating the erythropoiesis. The serum TfR was increased in iron deficiency anemia, hemolytic anemia and polycythemia and was decreased in aplastic anemia. In renal anemia, it was increased after the administration of erythropoietin (Epo). By the in vitro liquid culture of peripheral blood stem cells using interleukin 3 and Epo, it was found that soluble TfR was derived from the erythroblasts during the maturation process.     

The usefulness of serum transferrin receptor for discriminating iron deficiency without anemia in children

We determined serum transferrin receptor (sTfR), serum erythropoietin and hematologic and biochemical iron parameters in 251 healthy children. The levels of sTfR were significantly higher in children with storage iron deficiency but had a poor sensivity for recognizing iron deficiency without anemia. When ferritin values cannot accurately demonstrate the iron deficiency in children, the sTfR/ferritin ratio or sTfR-log ferritin is recommended to discriminate iron deficiency in the absence of anemia.     

Erythroblast iron metabolism in sideroblastic and sideropenic states

Iron appears to exert self-regulatory control over erythroblast iron uptake, iron storage and its incorporation into haem. It does this via iron regulatory proteins (IRPs) which bind reversibly to the iron responsive elements (IREs) on the mRNA of transferrin receptor (TfR), erythroid 5-aminolaevulinic acid synthase (ALA-S2) and ferritin. Iron deficiency leads to the binding of IRP to IRE. This binding inhibits the translation of mRNA for ALA-S2 and ferritin but stabilizes mRNA for TfR expression. Sideroblastic erythropoiesis is highly ineffective and characterized by mitochondrial iron loading. The study of X-linked sideroblastic anaemia has shown that the entry of iron into the mitochondria is poorly controlled and able to occur when protoporphyrin production is reduced, as is seen with the ALA-S2 mutations, or when it is increased as has been seen with ABC7 transporter mutations. Sideropenia characterises both iron deficiency anaemia (IDA) and the anaemia of chronic disease (ACD). Erythroblasts in ACD seem doubly equipped to protect their iron supply with their ability to increase the efficiency of transferrin-iron uptake as well as to activate the IRP/IRE system to increase surface TfR production. This increase in efficiency restricts the need to increase surface TfR production and maintains serum soluble TfR (sTfR) values within the normal range in iron replete ACD. The coexistence of iron deficiency with chronic disease, however, is associated with an increase in both the efficiency and number and a highly significant rise in sTfR values.     

Serum soluble transferrin receptor and the prediction of marrow aspirate iron results in a heterogeneous group of patients

Serum soluble transferrin receptor (sTfR) concentration has been evaluated in the diagnosis of iron deficiency in otherwise healthy individuals and in patients with rheumatoid arthritis, but has not been studied in a general population of patients with complicated clinical presentations. In this study, 145 anaemic patients with a variety of medical conditions undergoing diagnostic bone marrow aspiration for any reason were tested by a complete blood count, a panel of biochemical tests to evaluate iron status, bone-marrow aspirate iron stain, and serum sTfR concentration. Sixteen per cent lacked stainable iron in the marrow aspirate. All biochemical parameters differed significantly between patients with or without stainable marrow iron. The sTfR assay was significantly more sensitive but less specific than other iron status assays in identifying the absence of stainable iron. Logistic regression analysis demonstrated that only sTfR and ferritin contributed independently to the prediction of marrow iron status. Serum ferritin alone was highly specific but insensitive. A decision algorithm combining serum ferritin and sTfR was as sensitive as TfR and as specific as serum ferritin. The measurement of serum sTfR, especially in conjunction with serum ferritin, is a valuable addition to the existing methods for predicting the results of marrow aspirate iron stains.     

Serum transferrin receptor

Transferrin receptors (TfRs) are the conventional pathway by which cells acquire iron for physiological requirements. Under iron-deficient conditions there is an increased concentration of surface TfR, especially on bone marrow erythroid precursors, as a mechanism to sequester needed iron. TfRs are also present in the circulation, and the circulating serum TfR (sTfR) level reflects total body TfR concentration. Under normal conditions erythroid precursors are the main source of sTfR. Disorders of the bone marrow with reduced erythroid precursors are associated with low sTfR levels. The sTfR concentration begins to rise early in iron deficiency with the onset of iron-deficient erythropoiesis, and continues to rise as iron-deficient erythropoiesis progressively worsens, prior to the development of anemia. The sTfR level does not increase in anemia of chronic inflammation, but is increased when anemia of chronic inflammation is combined with iron deficiency. The sTfR level is also increased in patients with expanded erythropoiesis, including hemolytic anemias, myelodysplastic syndromes, and use of erythropoietic stimulating agents. The ratio of sTfR/ferritin can be used to quantify the entire spectrum of iron status from positive iron stores through negative iron balance, and is particularly useful in evaluating iron status in population studies. The sTfR/log ferritin ratio is valuable for distinguishing anemia of chronic inflammation from iron deficiency anemia, whether the latter occurs alone or in combination with anemia of chronic inflammation.     

Factor V Leiden mutation carriership and venous thromboembolism in polycythemia vera and essential thrombocythemia

The aim of the present study is to evaluate in an elderly hospitalized population the diagnostic value of the serum transferrin receptor (sTfR) in distinguishing IDA (iron deficiency anemia) from ACD (anemia of chronic disease) as compared to conventional laboratory tests of iron metabolism, especially serum ferritin. In a prospective study, 34 patients with IDA and 38 patients with ACD (a chronic disorder in 23 and an acute infection in 15) were evaluated using iron status tests including serum transferrin receptor assay. The iron stores were assessed by bone marrow examination. sTfR levels were elevated (>28.1 nmol/L) in 68% of the IDA patients but also in 43% of the patients with ACD-chronic inflammation and 33% with ACD-acute infection. Serum ferritin was the best test to differentiate IDA from ACD patients. We conclude that serum ferritin is a more sensitive and specific parameter than the sTfR assay to predict the bone marrow iron status in an elderly anemic population.     

Ret-Y a measure of reticulocyte size: a sensitive indicator of iron deficiency anemia

In this study the size of reticulocytes was measured, reticulocyte-Y (Ret-Y), to distinguish iron deficiency anemia from the anemia of chronic disease using a Sysmex XE2100 cell counter. We evaluated this parameter prospectively in 100 patients seen for the evaluation of anemia. A clinical diagnosis of iron deficiency anemia or anemia of chronic disease was made on the basis of a complete blood count, examination of the peripheral smear, and serum ferritin along with a history and physical examination. We analyzed the sensitivity and specificity of the Ret-Y in relationship to the clinical diagnosis. We also measured serum transferrin receptor levels to use as the gold standard laboratory test for iron deficiency against which we compared the Ret-Y. In 40 normal individuals with normal serum ferritin and transferrin receptor levels the mean Ret-Y was 1874 +/- 178 (1 SD). The mean Ret-Y in the anemia of chronic disease group (n=62) was 1722 +/- 162, not significantly different from normal. The mean Ret-Y value among iron-deficient patients (n=38), was 1407 +/- 136 (P <0.01 vs. the anemia of chronic disease group's Ret-Y value). Receiver operator curves showed that Ret-Y correlated closely to the serum transferrin receptor and was superior to the mean corpuscular volume, and ferritin level, in differentiating the type of anemia. The Ret-Y parameter has the highest overall sensitivity and specificity of the panel of tests routinely used in differentiating iron deficiency anemia from anemia of chronic disease.     

Heterotypic interactions between transferrin receptor and transferrin receptor 2

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.     

Iron status in pregnant women: which measurements are valid?

Anaemia in pregnancy in developing countries continues to be a public health problem of significant proportion. At least 50% of the anaemia has been blamed on iron deficiency. In populations where chronic inflammation and iron deficiency anaemia coexist, the criteria to accurately define iron status are not always clear. Similarly, in pregnancy, with marked physiological changes, cut-off points for biochemical parameters need to be re-examined. In this study we examined the diagnostic accuracy of iron parameters including mean cellular volume (MCV), serum iron, transferrin, total iron binding capacity (TIBC) and its saturation, zinc protoporphyrin (ZPP), ferritin and serum transferrin receptor (TfR) for the assessment of iron status in a population of anaemic pregnant women in Malawi. Stained bone marrow aspirates were used as the standard for comparison.
Results show that for the purpose of screening, serum ferritin is the best single indicator of storage iron provided a cut-off point of 30 μg/l is used. A number of other commonly used parameters of iron status were shown to have limited diagnostic accuracy. Logistic regression was used to obtain mathematical models for the prediction of bone marrow iron status using a combination of available parameters.     

Soluble transferrin receptor and soluble transferrin receptor/log ferritin index in diagnosis of iron deficiency anemia in pediatric inflammatory bowel disease

Background

There is no single reliable marker of iron homeostasis in inflammatory bowel disease.

The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non-Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.     

Microcytic and hypochromic anemias

In the majority of cases, microcytosis is the result of impaired hemoglobin synthesis. Disorders of iron metabolism and protoporphyrin and heme synthesis, as well as impaired globin synthesis, lead to defective hemoglobin production and to the generation of microcytosis and microcytic anemia. Iron deficiency anemie, anemia of chronic diseases, thalassemias, congenital sideroblastic anemias and homozygous HbE disease are the main representatives of microcytosis and microcytic anemias. Serum iron, total iron binding capacity, transferrin saturation, serum ferritin, serum transferrin receptor, transferrin receptor-ferritin index, and zinc-protoporhyrin concentration in erythrocytes are tests used for assessment of iron deficiency. The convention laboratory test for diagnosing iron deficiency is the measurement of serum ferritin. The most precise method for evaluating body iron stores is the examination for iron on aspirated bone marrow or marrow biopsy. Increased content of Hb A2 over 3.5% is diagnostic for beta-thalassemia. Presence of ringed sideroblasts is characteristic of sideroblastic anemias. Hemoglobin electrophoresis is required for the diagnosis of hemoglobinopathy E. The optimal therapeutic regimen in iron deficiency anemia used in this country is to administer 100 mg of elemental iron twice daily separately from meals. Ferrous sulphate (Ferronat Retard tbl. or Sorbifer Dulures tbl.) which are slow-releasing iron formulations are preferred because of their low cost, high bioavailability and low side-effects. Parenteral iron therapy is justified only in patients who cannot absorb iron, who have blood losses that exceed the maximal absorptive capacity of their intestinal tract or who are totally intolerant of oral iron. However, parenteral iron therapy may be associated with serious and even fatal side-effects.     

Soluble transferrin receptor in Aboriginal children with a high prevalence of iron deficiency and infection

OBJECTIVES: Aboriginal children in tropical Australia have a high prevalence of both iron deficiency and acute infections, making it difficult to differentiate their relative contributions to anaemia. The aims of this study were to compare soluble transferrin receptor with ferritin in iron deficiency anaemia (IDA), and to examine how best to distinguish the effect of iron deficiency from infection on anaemia. METHODS: We conducted a prospective study of 228 admissions to Royal Darwin Hospital in children from 6 to 60 months of age. Transferrin receptor concentrations were measured by a particle-enhanced immunoturbidimetric assay and ferritin by a microparticle enzyme immunoassay. RESULTS: On multiple regression, the best explanatory variables for haemoglobin differences (r2=33.7%, P<0.001) were mean corpuscular volume (MCV), red cell distribution width (RDW) and C-reactive protein (CRP); whereas transferrin receptor and ferritin were not significant (P>0.4). Using > or =2 abnormal indices (MCV, RDW, blood film)+haemoglobin <110 g/l as the reference standard for IDA, transferrin receptor produced a higher area under the curve on receiver operating characteristic curve analysis than ferritin (0.79 vs. 0.64, P<0.001) or the transferrin receptor-ferritin index (0.77). On logistic regression, the effect of acute infection (CRP) on haemoglobin was significant (P<0.001) at cut-offs of 105 and 110 g/l, but not at 100 g/l when only iron deficiency indicators (MCV, RDW, blood film) were significant. CONCLUSIONS: Transferrin receptor does not significantly improve the diagnosis of anaemia (iron deficiency vs. infection) over full blood count and CRP, but in settings with a high burden of infectious diseases and iron deficiency, it is a more reliable adjunctive measure of iron status than ferritin.     

Expression of cell surface transferrin receptor and intracellular ferritin after in vitro stimulation of peripheral blood T lymphocytes

Measurements of peripheral blood lymphocyte surface transferrin receptor using flow cytometry show that phytohaemagglutinin (PHA) stimulation causes a marked increase in both the number of cells bearing receptors and the absolute number of receptors. This increase is accompanied by the interleukin-2 receptor and there is a progressive increase of cells in the S phase of their cycle. Furthermore, ferritin synthesis in proliferating cells, as determined by immunoprecipitation techniques, increases significantly compared to non-stimulated cells. Intracellular concentrations of both spleen-type and heart-type ferritin are also increased with a preferentially high proportion of heart-type ferritin. These results suggest that expression of transferrin receptor and synthesis of ferritin is regulated by a complex mechanism. Cellular proliferative activity increases the expression of transferrin receptor and could modulate the biosynthesis of intracellular ferritin that is normally controlled by iron. These two processes do not appear to be linked.