The Role of Iron in the Regulation of Hepatic Transferrin Synthesis

The role of iron supply in the regulation of hepatic transferrin synthesis by the isolated perfused rat liver was studied using nutritional iron deficiency as the experimental model. The increased transferrin release encountered in iron deficiency could be equated with enhanced de novo synthesis as evidenced by the inhibitory effects of cycloheximide and measurements of intrahepatic protein pools before and after perfusion. Refeeding with iron, sufficient to restore plasma iron and hepatic ferritin iron but before correction of anaemia, promoted a reduction towards normal in the transferrin synthetic rate. This effect was not produced by transfusional correction of the anaemia, suggesting a specific response to iron supply. Phenobarbitone treatment, which produced a marked fall in hepatic ferritin iron concentration but no change in haemoglobin or plasma iron concentrations, promoted a specific enhancement of transferrin synthesis in both control and iron deficient livers. The concentration of liver iron stores appears to be a major regulatory factor in the control of hepatic transferrin synthesis.     

Hepatic Xanthine Oxidase and Ferritin Iron in the Developing Rat

The absence of hepatic xanthine oxidase in the fetus and newborn rat isassociated with a very high liver ferritin iron content. Soon after birth hepaticxanthine oxidase activity increases significantly coincident with a markeddecrease in liver ferritin iron content. At weaning, hepatic ferritin iron isvery low but slowly rises subsequent to intake of a normal diet containingiron.

Submitted on September 2, 1964 Accepted on November 11, 1964     

Studies on Congenital Hemolytic Syndromes. IV. Gastrointestinal Absorption of Iron

1. Results of studies of gastrointestinal absorption of ferrous iron in normalchildren and those with heterozygous thalassemia were similar.

2. In one patient with absent erythropoiesis but severe anemia, no increasein the amount of iron absorbed was noted.

3. In sickle cell-hemoglobin C disease and hereditary spherocytosis havingonly slight anemia in the presence of increased erythropoiesis, normal amountsof iron were absorbed.

4. Patients with sickle cell anemia and thalassemia major in whom therewas active erythropoiesis and marked anemia absorbed abnormally largeamounts of iron. The amount absorbed by individuals with the latter diseasecould be reduced by administration of transfusions and concomitant suppression of erythropoiesis.

5. Usual values for serum iron and latent iron-binding capacity in severalcongenital hemolytic syndromes have been presented and their significancediscussed.

6. No specific effect on absorption was noted by increased or reducedamounts of tissue or serum iron or by reduced or increased latent iron-bindingprotein.

Submitted on June 20, 1961 Accepted on October 30, 1961     

The Control of Hepatic Iron Uptake: Correlation with Transferrin Synthesis

S ummary . The control of hepatic iron uptake was studied in the perfused liver isolated from rats subjected to nutritional iron deficiency. The total hepatic iron uptake and incorporation into ferritin was found to be higher in iron deficiency and during the 48 h of oral refeeding with iron than in the normal state. Specific incorporation of iron into ferritin from a perfusate of normal transferrin iron saturation was enhanced in nutritional iron deficiency as compared to controls after 5 h of perfusion but not after 1 h, suggesting that increased uptake of iron from the perfusate may play a role in stimulating hepatic ferritin synthesis and assembly. This promotion of uptake into ferritin was inhibited by cycloheximide suggesting that enhanced incorporation of iron is dependent upon de novo synthesis of apoferritin. In control, nutritionally iron deficient and iron-refed rats there was a significant, direct correlation between the transferrin-iron saturation of the perfusate at physiological transferrin concentrations and total hepatic iron uptake after 5 h perfusion.
A significant positive correlation was found between the hepatic total and ferritin iron uptake and the transferrin synthetic rate measured in the same liver. It is proposed that in the liver the negative feedback of iron supply on transferrin synthesis may be linked with a positive feedback on ferritin synthesis. The timecourse of these reciprocal responses suggests a role for hepatic ferritin and/or a component of the non-haem, non-ferritin iron pool in the regulation of transferrin synthesis.     

The Regulation of Iron Release from the Perfused Rat Liver

Summary Factors affecting iron efflux from the isolated perfused rat liver were studied following the intravenous administration of transferrin-⁵⁹Fe or transferrin-⁵⁵Fc administered to the rat from 1·5 h to 3·5 d before perfusion of the liver. The liver was perfused with rat red cells suspended either in rat plasma or Eagle's Basal Medium (EBM). The mean rate of efflux into a plasma pool containing normal iron and transferrin concentrations was 0·9% of the initial hepatic radioactive iron pool per hour. In EBM the average rate of efflux was 0·1%/h and this could be increased to the rate observed with plasma by the addition of apotransferrin. The rate of iron release from the liver in the presence of apotransferrin or other chelators was inversely proportional to the time of prelabelling. Maximal release rates were observed in livers perfused within 5 h of administering transferrin-⁵⁹Fe to the rat. The effect of apotransferrin on efflux into EBM was concentration dependent. However, the maximum release of liver iron by apotransferrin occurred at physiological apotransferrin concentrations and addition of apotransferrin to plasma produced no increase in the rate of iron efflux. The stimulation of iron release in EBM caused by apotransferrin could be reversed by reducing the unsaturated iron binding capacity of the perfusate, either by addition of iron or removal of apotransferrin. However, increasing the iron concentration in the perfusate by the addition of iron-saturated transferrin without any reduction in the unsaturated iron binding capacity additionally increased iron release into plasma and EBM. This presumably reflects the exchange of plasma transferrin-³⁶Fe for liver ⁵⁹Fe. Hence iron release measured in these studies represents the sum of two processes—net release of ⁵⁹Fe induced by apotransferrin and iron exchange between plasma and liver iron pools. Apotransferrin and desferrioxamine were equally effective, per unit iron binding capacity, in mobilizing liver iron, and may compete for the same parenchymal iron pool. This suggests that mobilization of iron by apotransferrin may depend solely onits ability to chelate ferric iron and not on a more specific ferroxidase activity or interaction with membrane receptors.     

Hepatic Iron Quantitation and Its Relationship with Disease Measures and Histologically Assessed Iron Content

Purpose The study was undertaken to determine the relationships between the histological finding of hepatic injury, necrosis, fibrosis and iron staining with biochemical measures of hepatic injury and hepatic iron content. Methods One hundred fourteen consecutive subjects undergoing liver biopsy were studied. Data pertaining to biopsy reports, histology activity index, hepatic iron concentration, hepatic iron index, and histological grading of iron were recorded and analyzed using regression analysis. Results Statistical relationships were found between the hepatic iron index, histological grading of iron and hepatic iron concentration, whereas histology activity index was related to hepatic grading of iron, but not to hepatic iron concentration. Conclusions The pathologist’s assessment of the histological grading of iron relates directly to the histology activity index and hepatic iron concentration. Serum measures of iron, total iron-binding capacity and ferritin can be used to monitor hepatic iron content more economically and simply than with hepatic iron concentration measurement on liver biopsy.     

Studies in Iron Metabolism. IV. Iron Absorption in Experimental Iron Deficiency

1) Whole body counting by means of a large phosphor well scintillationcounter has been used to measure the absorption of Fe⁵⁹-tagged inorganic iron,and shown to compare favourably with other methods.

2) There is a delay in the fecal elimination of the unabsorbed portion ofthe dose of Fe⁵⁹ by iron-deficient rats on iron-deficient diet. The cause of thisdelay is unknown but it may be associated with the marked cecal enlargementwhich exists in these animals.

3) It is confirmed that iron deficiency is associated with striking enhancement of absorption of ferrous and ferric inorganic iron.

4) When a series of doses of ferrous iron of increasing size from 5 to 1,000µg. was given, there was a progressive increase in absorption for each increasein dose in both iron-supplemented and iron-deficient rats. The relationshipbetween amount of iron given and amount absorbed suggests that two processesmay be involved: 1) simple diffusion, and 2) a carrier mechanism.

5) The effect on iron absorption of a sudden change in iron intake hasbeen investigated. Switch from a low to high iron diet reduces absorption,and from a high to a low iron diet increases absorption, too rapidily for hemoglobin level or body iron stores alone to be the most important governingfactors and this finding emphasizes the importance of local changes in theintestine.

Submitted on April 23, 1962 Accepted on June 25, 1962     

Acute intestinal iron intoxication. I. Iron absorption,serum iron and autopsy findings

To simulate the fatal iron poisoning reported in children and to study themechanism of the iron toxicity, dissociable iron salts were given by stomach orduodenal tube or by enema to rabbits and dogs. In either route of administration, the lethal dose was found to be approximately 150 to 200 mg. Fe per kilogram body weight.

Dissociable iron salts in toxic doses were rapidly absorbed through the histologically intact mucosa both from the small and from the large bowel. In themajority of the animals, no histological changes were seen in the intestinalmucosa, but intestinal bleeding occurred in some instances by diapedesis fromthe greatly congested capillaries.

Serum iron levels of several milligrams per cent developed within sixty minutes after ingestion of the iron salts, and with the exception of 300 to 400 micrograms per cent, the serum iron was non-beta₁-globulin bound and in ferric state.

Although only a fraction of the total dose administered was found to be absorbed within nine hours, the observed serum iron rise was roughly proportionalto the dose ingested, and the survival time varied inversely with the dose.

Acute intestinal iron poisoning must therefore be considered as a true absorptive intoxication. Any possibly occurring mucosal damage in stomach andintestines is of secondary importance.

Submitted on March 22, 1954 Accepted on July 6, 1954     

Iron overload across the spectrum of non‐transfusion‐dependent thalassaemias: role of erythropoiesis,splenectomy and transfusions

Non‐transfusion‐dependent thalassaemias (NTDT ) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with β thalassaemia intermedia (n  = 95), haemoglobin (Hb) E/β thalassaemia (n  = 49) and Hb H syndromes (n  = 22). Liver iron concentration (LIC ), serum ferritin (SF ), transferrin saturation (TfSat) and non‐transferrin‐bound iron (NTBI ) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR ), labile plasma iron (LPI ) and nucleated red blood cells (NRBC s), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI , TfSat, NRBC s and SF relative to LIC , than non‐splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR , growth differentiation factor‐15, NRBC s) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI , LPI and NRBC s. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra‐hepatic iron distribution are more likely in previously transfused, splenectomised and iron‐overloaded NTDT patients with TfSat >70%.     

Mechanisms of plasma non‐transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients

In transfusional iron overload, extra‐hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non‐transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond‐Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin‐iron utilization (DBA).     

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/b2 globin gene deletion) mice

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb^th3/+ mice. Mice with the Hbb^th3/+ phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb^th3/+ mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.     

Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis.

Genetically hypotransferrinemic mice accumulate iron in the liver and pancreas. A similar pattern of tissue iron accumulation occurs in humans with hereditary hemochromatosis. In both disorders, there is a decreased plasma concentration of apotransferrin. To test the hypothesis that nontransferrin-bound iron exists and is cleared by the parenchymal tissues, the tissue distribution of 59Fe was studied in animals lacking apotransferrin. Two groups of animals were used: normal rats and mice whose transferrin had been saturated by an intravenous injection of nonradiolabeled iron, and mice with congenital hypotransferrinemia. In control animals, injected 59Fe was found primarily in the bone marrow and spleen. In the transferrin iron-saturated animals, injected 59Fe accumulated in the liver and pancreas. Gastrointestinally absorbed iron in hypotransferrinemic or transferrin iron-saturated mice was deposited in the liver. This indicates that newly absorbed iron is released from mucosal cells not bound to transferrin. Clearance studies demonstrated that transferrin-bound 59Fe was removed from the circulation of rats with a half-time of 50 min. In transferrin iron-saturated animals, injected 59Fe was removed with a half-time of less than 30 s. Analysis of the distribution of 59Fe in serum samples by polyacrylamide gel electrophoresis demonstrated the presence of 59Fe not bound to transferrin. These results demonstrate the existence of and an uptake system for non-transferrin-bound iron. These observations support the hypothesis that parenchymal iron overload is a consequence of reduced concentrations of apotransferrin.     

Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload

BACKGROUND/AIMS: Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS: Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS: No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS: Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).     

Uptake of Transferrin-Bound Iron by Rat Cells in Tissue Culture

The in vitro uptake of transferrin-bound iron by rat liver cells and rat embryo cells in culture, and by rat reticulocytes, was studied using labelled rat serum. The uptake of iron by rat liver cells was linear with time and showed a curvilinear relationship with the serum iron concentration. Over 40% of the cell radioactivity was found to be ferritin associated. Incubation of rat transferrin doubly labelled with ⁵⁹Fe and ¹²⁵l showed a difference between the uptakes of the two isotopes suggesting reflux of transferrin from the cell after iron uptake. In order to compare iron uptake from half saturated (Tf-Fe 1) and fully saturated (Tf-Fe2) transferrin, the three cell systems were each incubated in sera labelled with ⁵⁵Fe at 10% saturation and with ⁵⁹Fe at varying iron saturations ranging from 10% to 90%. Similar experiments were performed with sera having been randomly labelled with ⁵⁵Fe and labelled with ⁵⁹Fe but preincubated with reticulocytes in order to produce iron bound to non-reticulocyte orientated transferrin binding sites. The data obtained confirms the presence of a functional heterogeneity with respect to the half and fully saturated transferrin. Thus iron uptake from fully saturated transferrin was almost twice that for the half saturated molecule with rat liver cells or reticulocytes, and over three times that of the half saturated molecule with rat embryo cells. In contrast no such differences could be detected when iron uptake from serum randomly labelled with iron and serum with labelled non-reticulocyte orientated binding sites were compared. Finally, the effect of pre-incubating each of the three cell systems in unlabelled sera of varying percentage iron saturations on subsequent radio-iron uptake was studied. An inverse relationship between percentage saturation of the preincubation serum and subsequent uptake was demonstrated indicating that cellular mechanisms also influence cell iron uptake.     

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.     

Transferrin Synthesis in the Rat: A Study Using the Fluorescent Antibody Technique

The fluorescent antibody technique has been used to investigate transferrin synthesis in rats. The alteration in hepatic transferrin content has been noted in rats suffering from acute blood loss, chronic iron deficiency before and after treatment with iron, iron loading and localized hepatic sepsis. Marked changes in the transferrin content in the rat liver can be correlated with changes in erythropoietic rate, plasma transferrin concentration and the associated variation in catabolic rate of transferrin in these different situations. Rapid increases in hepatic transferrin content are considered to represent a synthetic response and are associated with elevation of plasma transferrin concentration. The synthesis response follows an extreme reduction in liver transferrin content; after acute blood loss and treatment of chronic iron deficiency anaemia with iron, a transferrin synthesis response coincides with the onset of erythropoiesis. The injection of iron-dextran into normal rats appears to be associated with a suppression of transferrin synthesis during the period of peak hepatic ferritin formation. It is thought that iron influences transferrin synthesis through its regulatory effect on erythropoietic rate.     

Absorption of Hemoglobin Iron: The Role of Xanthine Oxidase in the Intestinal Heme-Splitting Reaction

Heme from ingested hemoglobin—⁵⁹Fe is taken into the epithelial cell ofthe small intestinal mucosa of the dog and the ⁵⁹Fe subsequently appears inthe plasma bound to transferrin. A substance was demonstrated in homogenates of the mucosa which releases iron from a hemoglobin substrate invitro. Thus: (1) The addition of catalase to the mucosal homogenate reducesthe "heme-splitting" reaction. In contrast, sodium azide, a catalase inhibitor,potentiates the reaction. This suggests that a peroxide generating systemparticipates in the "heme-splitting" reaction. (2) Xanthine oxidase, an enzyme present in the intestinal epithelial cell, produces H₂O₂ by oxidation ofits substrate. The addition of allopurinol, a xanthine oxidase inhibitor, to theintestinal mucosal homogenate diminishes the "heme-splitting" reaction. (3)Fractionation of the 50,000 Gm. supernatant of the mucosal homogenate on aG-200 Sephadex column shows the "heme-splitting" activity to have the sameelution volume as xanthine oxidase, indicating a similar molecular weight. (4)The addition of a mucosal homogenate to a xanthine substrate results in theproduction of uric acid. These data suggest that xanthine oxidase in the intestinal epithelial cell is important in the release of iron from absorbed heme.The enzyme mediates the "heme-splitting" reaction by the generation ofperoxides which, in turn, oxidize the alpha-methene bridge of the heme ringreleasing iron and forming biliverdin.

Submitted on May 23, 1969 Accepted on July 31, 1969     

Effects of Inflammation on Iron and Transferrin Metabolism

S ummary . Experimentally induced acute inflammation in rats is associated with a fall in plasma iron and iron-binding capacity. Transferrin synthesis by the liver of these animals is normal and not increased in the presence of a low serum iron, as it is in iron deficiency.
Human splenic macrophages and rabbit pulmonary macrophages which were maintained in culture for up to 1 week showed an ability to take up protein-bound iron and labelled plasma proteins into the cytoplasm.
It is suggested that the macrophage may be the site both of iron sequestration and of transferrin degradation. This would provide a unified hypothesis to account for the finding of a low serum iron and TIBC in inflammatory disorders.     

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.