Deficiency Transferrin Research Articles

Gastrointestinal iron excretion and reversal of iron excess in a mouse model of inherited iron excess.

The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

The use of hypotransferrinemic mice in studies of iron biology.

The hypotransferrinemic (hpx) mouse is a model of inherited transferrin deficiency that originated several decades ago in the BALB/cJ mouse strain. Also known as the hpx mouse, this line is almost completely devoid of transferrin, an abundant serum iron-binding protein. Two of the most prominent phenotypes of the hpx mouse are severe anemia and tissue iron overload. These phenotypes reflect the essential role of transferrin in iron delivery to bone marrow and regulation of iron homeostasis. Over the years, the hpx mouse has been utilized in studies on the role of transferrin, iron and other metals in a variety of organ systems and biological processes. This review summarizes the lessons learned from these studies and suggests possible areas of future exploration using this versatile yet complex mouse model.

Reversibility of Iron Loading in Transferrin-Deficient Mice

An abundant serum metal-binding protein, transferrin is a critical factor in mammalian iron homeostasis. As transferrin is essential for iron delivery for erythropoiesis, transferrin-deficient patients and mice develop anemia but also a paradoxical systemic iron overload. Iron overload results from deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Using transferrin-deficient mice, we have previously shown that transferrin deficiency leads to hepcidin deficiency via two mechanisms. First, the anemia and/or hypoxia that results from inadequate iron delivery to bone marrow actively suppresses hepcidin expression by the liver. Second, transferrin directly stimulates hepcidin expression by the liver independently of transferrin's role in erythropoiesis; this second mechanism is dependent upon hemojuvelin, a bone morphogenetic protein coreceptor essential for hepcidin expression that is mutated in juvenile hemochromatosis. Here we present data examining the reversibility of iron loading in transferrin deficiency. Measurement of tissue metal levels in adult transferrin-deficient mice demonstrated progressive and systemic iron loading yet minimal to moderate changes in levels of other metals. Treatment of adult transferrin-deficient mice with exogenous transferrin normalized red cell parameters and hepcidin levels and reduced iron load in most organs. To further investigate the reduced tissue iron burden in transferrin-treated mice, we employed iron-loaded cell lines to demonstrate that cellular iron efflux can be stimulated by transferrin and that this efflux is specific to iron but not manganese or zinc. Current studies focus on two goals. First, we are determining if the reduction in tissue iron load represents solely a redistribution of iron stores to the erythron or additionally excretion of iron via urine and/or feces by measuring whole body iron levels in mice before and after transferrin treatment. Second, we are identifying transporters required for mobilization of iron stores using expression profiling of known and putative iron transporters in mouse tissues and validating the role of transporters of interest using our cell culture model. Results of these studies and their implications will be presented. Disclosures No relevant conflicts of interest to declare.

A Rare Case of Congenital Atransferrinemia: International Collaboration for Genetic Diagnosis

Congenital atransferrinemia is an extremely rare hematologic disease caused by transferrin deficiency, and characterized by microcytic hypochromic anemia with iron overload, and that can be fatal if left untreated. The prevalence is unknown. To date, there have been 16 reported cases from 14 families, with few cases confirmed genetically. Here, we report a new case from the Middle East that was diagnosed on clinical ground, and then confirmed by genetic testing through international collaboration.M.T is a 32 months old Egyptian boy, 2nd child of a consanguineous couple, with a healthy 5.5 years older sister. He was born at full term by SVD, suffered physiological jaundice as a newborn. He also had Talipes equinovarus and hypospadias.He was first brought to medical attention at a local hospital at the age of 8 months because of a febrile illness, when he was accidentally discovered to be very pale with Hb of 4.8 g/dl. He received packed RBC transfusion followed by oral iron therapy for 2 weeks, and then another assessment revealed Hb 6.9 g/dl, so he received another transfusion, and then referred to the University Hospital for further assessment.Examined at the age of 20 months, he had marked pallor, tachycardia, and mild hepatosplenomegaly, otherwise the child was normal with average anthropometric measurements.CBC revealed severe microcytic hypochromic anemia (Hb 4.8, MCV 54 fl, MCH 18 pg), MCHC 30 g/dl). Hb electrophoresis: normal Screening for Alfa and Beta thalassemia: normal wild type. Iron profile revealed hypoferremia (Serum iron: 6 ug/dl), TIBC: below assay range, and high serum ferritin: 669 ng/ml. Based on these results, atransferrinemia was suspected, so he was tested for serum transferrin, and reported as very low (8 mg/dl). Bone marrow aspirate was normocellular for age, showing normal granulopoiesis, normoblastic erythropoiesis, and normal megakaryopoiesis, no evidence of dysplasia or abnormal or malignant cells. Prussian blue stain showed depleted iron stores. Serum lead, bilirubin, liver and kidney function tests were normalBoth parents had normal blood picture and iron profile. Human apotransferrin was not available in Egypt, so, plasma transfusion was instituted every 2-4 weeks, through which regimen he is maintaining Hb level between 7-12 gm/dl.As molecular diagnosis for that very rare disease was not feasible in Egypt, after parental consent, blood samples from patient and both parents were sent to Dr. von Haunersches Kinderspital, Munich, and a novel transferrin genemutation was detected by NGS(TF, NM_001063.3, c.1247A>G, p.Y416C) that is currently being validated by Sanger sequencing.To the best of our knowledge, this is the first atransferrinemia report from the Middle East, where prevalence of thalassaemia and iron deficiency anemia is high. However, this rare anemia case underlines the importance of considering rare hereditary disorders like atransferrinemia as the cause for unexplained hypochromic microcytic anemia associated with iron overload, in order to initiate appropriate treatment and avoid iron overload related complications. DisclosuresNo relevant conflicts of interest to declare.

Investigating the role of transferrin in the distribution of iron, manganese, copper, and zinc.

The essential role of transferrin in mammalian iron metabolism is firmly established. Integral to our understanding of transferrin, studies in hypotransferrinemic mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery for erythropoiesis and in the regulation of expression of hepcidin, a hormone that inhibits macrophage and enterocyte iron efflux. Here we investigate a potential role for transferrin in the distribution of three other physiologic metals, manganese, copper, and zinc. We first assessed metal content in transferrin-rich fractions of wild-type mouse sera and demonstrate that although both iron and manganese cofractionated predominantly with transferrin, the absolute levels of manganese are several orders of magnitude lower than those of iron. We next measured metal content in multiple tissues in wild-type and hypotransferrinemic mice of various ages. Tissue metal imbalances were severe for iron and minimal to moderate for some metals in some tissues in hypotransferrinemic mice. Metal levels measured in a transferrin-replete yet hepcidin-deficient and iron-loaded mouse strain suggested that the observed imbalances in tissue copper, zinc, and manganese levels were not all specific to hypotransferrinemic mice or caused directly by transferrin deficiency. Overall, our results suggest that transferrin does not have a primary role in the distribution of manganese, copper, or zinc to tissues and that the abnormalities observed in tissue manganese levels are not attributable to a direct role for transferrin in manganese metabolism but rather are attributable to an indirect effect of transferrin deficiency on hepcidin expression and/or iron metabolism.

Transferrin Deficiency Leads To Specific and Partially Reversible Iron Overload

Studies in hpx mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery to the erythron and regulation of expression of hepcidin, a hormone that inhibits dietary iron absorption. Here we address two other putative roles for s transferrin. First, we determined if transferrin is essential for metabolism of metals other than iron. Metal content analysis of fractionated mouse sera indicated that iron was the most abundant metal in transferrin-rich fractions. Organ metal content analysis demonstrated severe imbalances for iron and minimal to moderate imbalances for copper, zinc and manganese in hpx tissues. Analysis of metal-dependent gene expression and organ metal content in a mouse model of inherited hepcidin excess suggested that hpx tissue imbalances in copper, zinc and manganese levels were not all physiologic or specific to primary transferrin deficiency. Second, we tested if transferrin modulates the mobilization of tissue iron stores. Transferrin treatment of hpx mice decreased iron levels in most tissues analyzed. Iron mobilization was independent of hepcidin, as iron levels decreased in hpx mice deficient in hemojuvelin, a membrane protein essential for hepcidin expression. Iron mobilization led to decreased ferritin iron and heavy chain levels. The decrease in liver ferritin iron stores did not match the decrease in total liver iron stores, suggesting that iron was mobilized from multiple storage sites. Overall, our analysis suggests that the primary consequences of transferrin deficiency are largely specific to iron and that transferrin can modulate the mobilization of tissue iron stores. Disclosures:No relevant conflicts of interest to declare.

Supportive therapy in medical therapy of head and neck tumors.

Fever during neutropenia may be a symptom of severe life threatening infection, which must be treated immediately with antibiotics. If signs of infection persist, therapy must be modified. Diagnostic measures should not delay treatment. If the risk of febrile neutropenia after chemotherapy is ≥20%, then prophylactic therapy with G-CSF is standard of care. After protocols with a risk of febrile neutropenia of 10–20%, G-CSF is necessary, in patients older than 65 years or with severe comorbidity, open wounds, reduced general condition. Anemia in cancer patients must be diagnosed carefully, even preoperatively. Transfusions of red blood cells are indicated in Hb levels below 7–8 g/dl. Erythropoiesis stimulating agents (ESA) are recommended after chemotherapy only when hemoglobin levels are below 11 g/dl. The Hb-level must not be increased above 12 g/dl. Anemia with functional iron deficiency (transferrin saturation <20%) should be treated with intravenous iron, as oral iron is ineffective being not absorbed. Nausea or emesis following chemotherapy can be classified as minimal, low, moderate and high. The antiemetic prophylaxis should be escalated accordingly. In chemotherapy with low emetogenic potential steroids are sufficient, in the moderate level 5-HT3 receptor antagonists (setrons) are added, and in the highest level Aprepitant as third drug.

Known and potential roles of transferrin in iron biology

Transferrin is an abundant serum metal-binding protein best known for its role in iron delivery. The human disease congenital atransferrinemia and animal models of this disease highlight the essential role of transferrin in erythropoiesis and iron metabolism. Patients and mice deficient in transferrin exhibit anemia and a paradoxical iron overload attributed to deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Studies of inherited human disease and model organisms indicate that transferrin is an essential regulator of hepcidin expression. In this paper, we review current literature on transferrin deficiency and present our recent findings, including potential overlaps between transferrin, iron and manganese in the regulation of hepcidin expression.

Transferrin-a modulates hepcidin expression in zebrafish embryos

AbstractThe iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a–dependent pathway in the zebrafish embryo.

Hereditary Hemochromatosis Protein, HFE, Interaction with Transferrin Receptor 2 Suggests a Molecular Mechanism for Mammalian Iron Sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.

Abnormal iron accumulation in the brain of neonatal hypotransferrinemic mice

Transferrin is a plasma protein involved in iron delivery to tissues. To study iron transport into the brain under a transferrin deficiency, iron concentration and 59Fe uptake in the brain were measured in neonatal hypotransferrinemic (HP) mice at 7 days of age. Brain iron concentration of the HP mice, in which iron concentration was relatively high in the cerebral cortex and cerebellum, was approximately three times higher than that of non-mutant mice, whereas serum iron concentration of HP mice was significantly lower than that of non-mutant mice. When 59FeCl 3 was subcutaneously injected into HP and non-mutant mice, 59Fe was distributed highly in the choroid plexus in the ventricles of HP mice 24 h after injection. The 59Fe distribution in the brain was different between HP and non-mutant mice. On the other hand, the clearance of 59Fe from the blood was very high in HP mice and the hepatic 59Fe concentration of HP mice was more than ten times of that of non-mutant mice. The present findings demonstrate that iron distribution in the brain is changed by transferrin deficiency and that iron abnormally accumulates in the brain of HP mice. It is likely that the management of iron is different in the brain of HP mice.

Importance of Anemia and Transferrin Levels in the Regulation of Intestinal Iron Absorption in Hypotransferrinemic Mice

AbstractThe hypotransferrinemic mouse (trf hpx) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass 59Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf hpxanimals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf hpx mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Importance of Anemia and Transferrin Levels in the Regulation of Intestinal Iron Absorption in Hypotransferrinemic Mice

The hypotransferrinemic mouse (trf hpx) is a mutant strain exhibiting transferrin deficiency, marked anemia, hyperabsorption of iron, and elevated hepatic iron stores. We set out to investigate the relative roles of anemia and of transferrin in the malregulation of intestinal iron absorption in these animals. Transfusion of erythrocytes obtained from littermate controls increased hemoglobin levels and reduced reticulocyte counts in recipient animals. Although mucosal to carcass 59Fe transfer was reduced, total duodenal iron uptake was not significantly affected. Iron absorption in homozygotes, in contrast to littermate controls, was not reduced by hyperoxia. Mouse transferrin injections, in the short term, increased delivery of iron to the marrow and raised hemoglobin levels. Although mucosal transfer and total iron uptake were reduced at the higher transferrin doses, total uptake was still higher than in controls. Daily injections of mouse/human transferrin for 3 weeks from weaning, normalized hemoglobin values, and markedly reduced liver iron and intestinal iron absorption values in trf hpxanimals. When such daily-injected mice were left for a week to allow transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron in trf hpx mice is not solely because of the anemia; transferrin levels per se do affect iron absorption, possibly via a direct effect on the intestinal mucosa.

Transferrin is an essential factor for myelination.

It has been established that oligodendrocytes, the myelin forming cells, participate in iron homeostasis through the synthesis and secretion of transferrin. Here we investigated whether a correlation exists between myelination, the commonly studied function of oligodendrocytes, and that of transferrin synthesis and secretion. We used a proteolipid protein mutant, the myelin deficient rat, whose condition is characterized by severe hypomyelination. We compared the ontogenic profile for transferrin gene expression in mutants with that of unaffected rat pups through northern blot analysis and in situ hybridization. Surprisingly, transferrin synthesis was null in mutant oligodendrocytes. Next, we demonstrated that a single apo-transferrin intraparenchymal injection administered to P5 rat pups enabled mutant oligodendrocytes to synthesize myelin basic protein and to myelinate axons, indicating that transferrin effects mutant oligodendrocyte maturation regardless of its source. Thus, transferrin availability is essential for oligodendrocyte maturation and function, and oligodendrocytes are most vulnerable to transferrin deficiency during the premyelinating stage.

Clinical pharmacokinetics of iron preparations.

The principle of iron conservation is the basis of iron metabolism; the normal basal loss of iron from the body is about 1 mg daily in a 70 kg man and 0.8 mg in a 55 kg woman. Iron is lost mainly by the menstrual and gastrointestinal routes. The total iron requirement during pregnancy is 800 mg; in the last month the requirement may amount to 7 to 8 mg/day. Supplementary iron is recommended for many menstruating women, and during the latter part of pregnancy. Correct fetal iron metabolism is ensured by proper maternal iron status, although there are contradictory opinions and findings about the relationship between maternal and fetal iron metabolism. Preterm infants fed on breast milk have a negative iron balance, and require an iron intake of about 0.6 mg/kg/day, and 3.4 mg/1 g haemoglobin, to compensate for intestinal and venesection iron losses, respectively. The absorption of supplementary iron by the preterm infant is a linear function of intake. Preterm infants do not require iron supplements when given repeated blood transfusions. During lactation the total iron losses of the mother are 1 mg/day, and thus no supplementary iron is needed if the iron metabolism has been in balance during the pregnancy. Serum ferritin concentration decreases continuously when iron stores in the body are reduced, and totally empty iron stores are the only known reasons for low serum ferritin concentration. Despite depleted iron stores, serum ferritin concentration can be normal or higher than normal in protein-energy malnutrition, up to 3 months after major surgery, in acute liver damage, in some patients with prolonged hyperglycaemia due to diabetes mellitus, in acute lobar pneumonia, active pulmonary tuberculosis and rheumatoid arthritis on gold therapy, in sepsis secondary to marrow hypoplasia induced by chemotherapy, in heavy drinkers and for a few days after myocardial infarction. In haemochromatosis, iron is deposited in liver (producing fibrosis), pancreas, endocrine glands and heart. The rise in the level of iron in the body is due to increased absorption and/or increased intake. This pathology may occur in transfusions, in alcoholism (especially when alcoholic beverages are contaminated with iron and the diet is low-protein), in several liver diseases, in congenital transferrin deficiency and in idiopathic disease. Patients susceptible to haemochromatosis should receive a low-iron diet. Serum ferritin determination may be helpful in early identification of susceptible members of a family with idiopathic familial haemochromatosis, but transferrin saturation is not a good indicator of either iron depletion or iron overload.(ABSTRACT TRUNCATED AT 400 WORDS)

A splicing defect in the mouse transferrin gene leads to congenital atransferrinemia

We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5′ and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.

A Splicing Defect in the Mouse Transferrin Gene Leads to Congenital Atransferrinemia

AbstractWe have analyzed the biochemical defect in a mutant line of mice that produces <1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are <20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5' and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.