Iron homeostasis in chronic inflammation

Inflammation induced anemia and resistance to erythropoietin are common features in patients with chronic kidney disease (CKD). Elevated levels of cytokines and enhanced oxidative stress, conditions associated with inflammatory states, are implicated in the development of anemia. Accumulating evidence suggests that activation of cytokine cascade and the associated acute-phase response, as it often occurs in patients with CKD, divert iron from erythropoiesis to storage sites within the reticuloendothelial system leading to functional iron deficiency and subsequently to anemia or resistance to erythropoietin. Other processes have also been shown to be involved in the pathogenesis of anemia provoked by the activated immune system including an inhibition of erythroid progenitor proliferation and differentiation, a suppression of erythropoietin production and a blunted response to erythropoietin. The present review concerns the underlying alterations in iron metabolism induced by chronic inflammation that result in anemia.     

Inflammation associated anemia and ferritin as disease markers in SLE

Introduction

In a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia.

Methods

A protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed.

Results

Protein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced.

Conclusion

Urine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE.     

Decoupling ferritin synthesis from free cytosolic iron results in ferritin secretion

Ferritin is a multisubunit protein that is responsible for storing and detoxifying cytosolic iron. Ferritin can be found in serum but is relatively iron poor. Serum ferritin occurs in iron overload disorders, in inflammation, and in the genetic disorder hyperferritinemia with cataracts. We show that ferritin secretion results when cellular ferritin synthesis occurs in the relative absence of free cytosolic iron. In yeast and mammalian cells, newly synthesized ferritin monomers can be translocated into the endoplasmic reticulum and transits through the secretory apparatus. Ferritin chains can be translocated into the endoplasmic reticulum in an in?vitro translation and membrane insertion system. The insertion of ferritin monomers into the ER occurs under low-free-iron conditions, as iron will induce the assembly of ferritin. Secretion of ferritin chains provides a mechanism that limits ferritin nanocage assembly and ferritin-mediated iron sequestration in the absence of the translational inhibition of ferritin synthesis.     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.     

BH3-only protein Noxa contributes to apoptotic control of stress-erythropoiesis

Apoptosis plays an essential role in the control of erythropoiesis under normal and pathological conditions. However, the contribution of individual proteins within cell death signalling pathways remains poorly defined. Here, we investigated the role of the pro-apoptotic Bcl-2 family member Noxa in the regulation of erythropoiesis. We found that expression of Noxa is induced during erythroid differentiation of human and murine precursor cells. Using in vitro model systems for erythroid progenitors, we observed rapid induction of Noxa upon cytokine deprivation. Knockdown or deletion of Noxa conferred significant protection against apoptosis upon cytokine withdrawal. In vivo, Noxa deficiency did not affect hematological blood parameters or erythroid progenitor composition of bone marrow and spleen under steady-state conditions. In contrast, in a model of acute haemolytic anemia, Noxa-deficiency enhanced hematocrit recovery. Moreover, in a model of chronic inflammation-induced anemia, Noxa-ablation resulted in a dramatic increase of erythroblast expansion. Our data indicate that induction of Noxa in erythroid progenitors sets a survival threshold that limits expansion beyond the number of cells that can be sustained by the available cytokines, which becomes apparent under conditions of induced anemia.     

The Effect of Iron Treatment on Adhesion Molecules in Patients with Iron Deficiency Anemia

The present study was aimed to determine the effect of iron supplementation on levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with iron deficiency anemia (IDA). In this study, 26 female patients diagnosed with iron deficiency were treated approximately 3 months of oral iron supplementation (99 ± 10 days; ferrous glycine sulfate; 100 mg/day of elemental iron). Levels of sICAM-1 and sVCAM-1 were assessed prior to treatment and after approximately 3 months of treatment and compared with 26 healthy female subjects. A significant increase in sVCAM levels was found in the patients with iron deficiency at the end of the treatment relative to pretreatment levels compared to controls, whereas no significant differences were determined in sICAM levels. In the posttreatment period, no significant change was observed in sICAM levels compared to the pretreatment levels, whereas sVCAM levels decreased. However, after the treatment period, the sVCAM, hemoglobin, mean corpuscular volume (MCV), and serum ferritin levels did not return to the normal range compared to the controls. Pretreatment sVCAM-1 levels were inversely correlated with levels of hemoglobin, hemotocrit, MCV, serum iron, and ferritin. After treatment, the sVCAM-1 levels were negatively correlated with ferritin levels. Levels of sVCAM were significantly higher in patients with IDA than controls. After the treatment period, the sVCAM levels were not completely normalized in patients with IDA compared to controls, regardless of the presence of inadequate levels of hemoglobin, MCV, and serum ferritin. Thus, iron supplementation not only ameliorates anemia, but may also reduce the inflammation markers in cases with IDA.     

Ferritin is not a required intermediate for iron utilization in heme synthesis

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.     

Ferritin heavy chain-mediated iron homoeostasis regulates expression of IL-10 in Chlamydia trachomatis-infected HeLa cells

Chlamydia trachomatis is the leading cause of sexually transmitted infection worldwide, in which disease outcome is determined by the balance between pro- and anti-inflammatory host immune responses. Iron plays important roles in regulation and enhancement of various pro- and anti-inflammatory cytokines. Earlier studies have established essentiality of iron in C. trachomatis infection; however, there is lack of study wherein modulatory effect of iron regulated protein [FHC (ferritin heavy chain)] in regulation of anti-inflammatory cytokine IL (interleukin)-10 has been investigated. In this study, immunoblotting results showed the up-regulation of FHC in C. trachomatis-infected HeLa cells in comparison with mock (in vitro control). Further secretory IL-10 level was significantly increased (P<0.001) or decreased (P<0.001) in response to iron supplementation [FAC (ferric ammonium citrate)] and depletion [DFO (deferoxamine)], respectively. However, in C. trachomatis-infected HeLa cells, levels of IL-10 remain higher, irrespective of availability of iron in comparison with their respective control. These results showed that secretion of IL-10 and expressions of FHC have concordance. Further, to understand interdependence of IL-10 and iron homoeostasis (regulation), the levels of IL-10 were compared with iron-responsive GFP (green fluorescent protein) expression in HeLa-229 cells. The mean fluorescent intensities of GFP were in accordance with levels of IL-10 in C. trachomatis-infected cells. These results showed the association of secreted IL-10, FHC and iron homoeostasis in C. trachomatis-infected HeLa-229 cells. This study provides insight into host-Chlamydia interaction at the crossroad of iron metabolism and immune responses and may help in realizing the potential of iron homoeostasis modulators in treatment of chronic chlamydial infection.     

Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

The effect of hemin in vitro and in vivo on human erythroid progenitor cells

Hemin stimulates erythropoiesis and hemoglobin synthesis in vitro. We cultured erythroid progenitor cells from normal individuals, patients with sickle cell anemia, and a patient with acute variegate porphyria who received intravenous hemin treatment, with 0-800 microM hemin added in vitro. Fifty to 200 microM hemin consistently stimulated colony growth from normal donors 2- to 8-fold, while concentrations of up to 400 microM were stimulatory in cultures from donors with sickle cell anemia. In vivo hemin decreased the number of blood BFU-e in the patient with porphyria, but did not abrogate the in vitro stimulatory effect of hemin. Hemin concentrations which increased colony numbers increased gamma globin synthesis in some studies and decreased it in others. Hemin thus has clearcut erythroid growth-potentiating activity, although a consistent effect on globin chain regulation is not apparent.     

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.     

Bone marrow clonogenic capability, cytokine production, and thymic output in patients with common variable immunodeficiency

In patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-alpha compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency.     

Severe malarial anemia associated with increased soluble Fas ligand (sFasL) concentrations in Gabonese children

To investigate if severe malarial anemia is associated with specific cytokine overproduction, we evaluated serum levels of soluble Fas ligand (sFasL), tumor necrosis factor (TNF-alpha) and interleukin-10 (IL-10) from three groups of young children with Plasmodium falciparum infection (asymptomatic cases, uncomplicated malaria cases and severe malarial anemia cases), in a hyperendemic area of Gabon. In uncomplicated cases, only TNF levels were significantly (p < 0.001) increased in comparison to asymptomatic cases with P. falciparum infection. High levels of sFasL, TNF-alpha and IL-10 were associated with low hemoglobin concentrations, sFasL levels were significantly higher in children with severe malarial anemia (p < 0.001) as compared to both other groups. The parasite density was positively correlated with IL-10, TNF-alpha and sFasL levels. TNF-alpha and sFasL, but not IL-10 or parasitemia, were independent predictors of hemoglobin concentrations. These results suggest that, in malaria, a specific dysregulation of the cytokine balance may lead to complications such as severe anemia.     

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.     

Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.     

UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis

UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and reactive oxygen species (ROS) modulation. High levels of UCP2 mRNA were recently found in erythroid cells where UCP2 is hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. We examined UCP2 protein expression and role in mice erythropoiesis in vivo. UCP2 was mainly expressed at early stages of erythroid maturation when cells are not fully committed in heme synthesis. Iron incorporation into heme was unaltered in reticulocytes from UCP2-deficient mice. Although heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of progenitor cells from bone marrow and fetal liver both in vitro and in vivo revealed that UCP2 deficiency results in a significant decrease in cell proliferation at the erythropoietin-dependent phase of erythropoiesis. This was accompanied by reduction in the phosphorylated form of ERK, a ROS-dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid cells revealed altered distribution of ROS, resulting in decreased cytosolic and increased mitochondrial ROS. Restoration of the cytosol oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.     

Is Obesity Associated With Anemia of Chronic Disease? A Population‐based Study

Obesity is characterized by chronic, low-grade, systemic inflammation, which, in turn, has been associated with anemia of chronic disease. We hypothesized that obesity may be associated with the features of anemia of chronic disease, including low hemoglobin concentration, low serum iron and transferrin saturation (TS), and elevated serum ferritin. We compared normal-weight to overweight and obese adult participants of the third National Health and Nutrition Examination Survey with respect to hemoglobin concentration and levels of serum iron, TS, and ferritin. Measured BMI was used to categorize participants into normal weight (BMI < 25 kg/m(2), n = 6,059), overweight (BMI 25 to <30 kg/m(2), n = 5,108), mildly obese (BMI 30 to <35 kg/m(2), n = 2,366), moderately obese (BMI 35 to <40 kg/m(2), n = 850), and severely obese (BMI > or = 40 kg/m(2), n = 465). After adjustment for age, gender, menstruation, race/ethnicity, education, alcohol consumption, smoking, blood donation, and dietary iron intake, serum ferritin was progressively higher with increasing BMI category, whereas serum iron and TS were progressively lower. However, compared to normal-weight persons, those in all other higher BMI categories did not have a significant change in hemoglobin concentration after adjustment for the above-mentioned confounders. Overweight and obesity were associated with changes in serum iron, TS, and ferritin that would be expected to occur in the setting of chronic, systemic inflammation. However, overweight and obese persons were not more likely to be anemic compared with normal-weight persons.     

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.     

Hyperferritinemia is associated with insulin resistance and fatty liver in patients without iron overload

Objective

During the last 10 years we have experienced an increasing number of referrals due to hyperferritinemia. This is probably due to increased awareness of hereditary hemochromatosis, and the availability of a genetic test for this condition. Most of these referred patients were over-weight middle-aged men with elevated ferritin levels, but without the hemochromatosis-predisposing gene mutations. We evaluated the relationship between hyperferritinemia and the metabolic syndrome in 40 patients.

Methods

Forty consecutive patients referred for hyperferritinemia were investigated. The examination programme included medical history, clinical investigation and venous blood samples drawn after an overnight fast. This resulted in 34 patients with unexplained hyperferritinemia, which were further examined. Liver biopsy was successfully performed in 29 subjects. Liver iron stores were assessed morphologically, and by quantitative phlebotomy in 16 patients.

Results

The majority of the patients had markers of the metabolic syndrome, and 18 patients (52%) fulfilled the IDF-criteria for the metabolic syndrome. Mean body mass index was elevated (28,8±4,2), mean diastolic blood pressure was 88,5±10,5 mmHg, and mean fasting insulin C-peptide 1498±539 pmol/l. Liver histology showed steatosis and nuclear glycogen inclusions in most patients (19 out of 29). Only four patients had increased iron stores by histology, of which two could be explained by alcohol consumption. Fourteen of 16 patients normalized ferritin levels after phlebotomy of a cumulative blood amount corresponding to normal iron stores. Ferritin levels were significantly related to insulin C-peptide level (p<0.002) and age (p<0.002).

Conclusion

The present results suggest that liver steatosis and insulin resistance but not increased iron load is frequently seen in patients referred for suspected hemochromatosis on the basis of hyperferritinemia. The ferritin level seems to be positively associated to insulin resistance.     

Iron mobilization in three animal models of inflammation

The effect of acute, subchronic, and chronic experimental models of inflammation upon hematocrit, hemoglobin, serum iron and ferritin iron and nonheme iron concentration in the liver and spleen has been studied in the rat. In the acute model (carrageenan oedema) no iron mobilization took place, whereas in the chronic models differences in iron mobilization were observed, related to their different chronicity and to the time elapsed from induction. The carrageenan-induced granuloma (from 12 h to 8 days) (subchronic model) was accompanied by a decrease of plasma iron (12 and 24 h), a later decrease of the hematocrit values (2 and 4 days) and high ferritin and nonheme iron concentrations in the liver and spleen for 4 days, followed by a tendency to return to the control values. The anemia in the adjuvant arthritis (from 1 to 4 weeks after induction) (chronic model) was observed at 7 days and is related to increased iron stores in the liver and spleen. However, the iron store levels in liver decreased and fell later below control values. The increase of ferritin and nonheme iron concentrations may be responsible for the reduced availability of iron release from tissue.