Ferritin Stores Research Articles

How does the ferritin core form?

Ferritin stores iron as ferrihydrite inside a shell composed of H and L protein chains. H chains contain ferroxidase centres catalysing Fe2+ oxidation, while L chains lack these centres but seem to promote ferrihydrite nucleation. Mossbauer spectroscopic studies of recombinant H-chain ferritins show the following: (1) fast Fe2+ oxidation is associated with the formation of Fe3+ dimers at the ferroxidase centre; (2) within 30 min, a portion of the dimers have split to Fe3+ monomers and some monomers have moved to the threefold channels; (3) over longer times, a trend dimer → monomer → core is established. Core formation is accelerated if L chains are present.

Coordmation of cellular iron metabolism by post-transcriptional gene regulation

Maintenance of cellular iron homeostasis demands the coordination of iron uptake, intracellular storage, and utilization. Recent investigations suggest that a single genetic regulatory system orchestrates the expression of proteins with central importance for all three aspects of cellular iron metabolism at the level of mRNA stability and translation. Two components of this regulatory system have been defined: a cis-acting mRNA sequence/structure motif called “iron-responsive element” (IRE) and a specific trans-acting cytoplasmic binding protein, here referred to as “IRE-binding protein” (IRE-BP). As an early event in the regulatory cascade, cellular iron deprivation induces the IRE-binding activity of IRE-BP, whereas binding activity is reduced in iron-replete cells. IRE-BP is highly homologous to the iron—sulphur (FeS) protein aconitase which strongly suggests that IRE-BP is an FeS protein itself. Control over IRE-BP activity by the cellular iron status is exerted post-translationally and likely involves changes between (4Fe-4S) and (3Fe-4S) states of the postulated IRE-BP FeS cluster. In addition, post-translational regulation of IRE-BP activity via heme has been proposed. Subsequent to its activation, IRE-BP binds with high affinity to single IREs contained in the 5′ untranslated regions (UTRs) of ferritin and erythroid 5-aminolevulinic acid synthase (eALAS) mRNAs. The binding represses translation of these proteins involved in iron storage and utilization, respectively. In contrast, iron uptake is largely regulated via multiple IREs in the 3′ UTR of transferrin receptor (TfR) mRNA. TfR-IREs are required for the iron-sensitive control of TfR mRNA stability. IRE-BP binding stabilizes TfR gene transcripts against as yet undefined ribonucleases. As a result of these regulatory interactions, iron starvation induces the expression of TfR, thereby increasing iron uptake, and represses the synthesis of proteins involved in iron storage and utilization. As cellular iron levels rise, the homeostatic balance is maintained by lowering iron uptake and increasing iron storage in ferritin.

Alveolar Macrophage Content of Isoferritins and Transferrin: Comparison of Nonsmokers and Smokers with and without Chronic Airflow Obstruction

Alveolar macrophages (AM) contain iron and ferritin, and concentrations of both are increased in AM of smokers compared with nonsmokers. Ferritin stores iron in a nontoxic form but can release iron in the presence of reducing agents and thereby catalyze the generation of toxic hydroxyl radicals via the Haber-Weiss reaction. Two distinct isoferritins are found in peripheral monocytes, L ferritin and H ferritin. H ferritin is the predominant isoferritin in human monocytes and is more effective than L ferritin in detoxifying iron in vitro. In this study we quantitated content of H and L ferritins, transferrin, and iron in AM recovered by bronchoalveolar lavage (BAL) of 24 subjects, including eight nonsmokers, eight smokers with normal spirometry, and eight smokers with chronic airflow obstruction (CAO). Of total AM ferritin in nonsmokers 95% was composed of L ferritin. Smokers without CAO demonstrated a 6.5-fold increase in the AM content of L ferritin (1,886 +/- 266 versus 290 +/- 51 ng, mean +/- SEM; p less than 0.0001) and a 3.8-fold increase in H ferritin (61 +/- 18 versus 16 +/- 2 ng per 1 x 10(6) AM, p less than 0.01) compared with nonsmokers. Compared with smokers without CAO, AM recovered from smokers with CAO demonstrated a greater increase in L ferritin (5,059 +/- 493 versus 1,886 +/- 266 ng per 1 x 10(6) AM, p less than 0.002) but a similar increase in H ferritin (64 +/- 8 versus 61 +/- 18 per 1 x 10(6) AM).(ABSTRACT TRUNCATED AT 250 WORDS)

Iron regulation of ferritin gene expression

Ferritin is a ubiquitous iron-storage protein found in the cells of animals, plants, molds, and bacteria which it protects from toxic intracellular levels of iron. Ferritin stores iron within a hollow protein shell formed by subunits of two types, H and L. The 5' untranslated regions of the two subunit mRNAs contain an almost identical 28-nucleotide sequence which regulates translation by binding to a specific cell sap protein. When cell iron level is low, this repressor protein obstructs translation of stored ferritin mRNAs, whereas increased iron levels release this protein, thus permitting extensive ferritin subunit synthesis to respond rapidly. Similar motifs in the 3' untranslated region of transferrin receptor mRNA interact with this protein to regulate breakdown of the mRNA and thus change the receptor population. Finally, transcription of the H and L genes can be independently increased by iron and other factors. In the case of iron, synthesis of the L-mRNA is increased preferentially since ferritin shells with a preponderance of L-subunits store iron more efficiently. Thus regulation of ferritin synthesis at the translational and transcriptional levels and by transferrin receptor mRNA abundance at the level of breakdown provide a coordinated mechanism for protecting cells against the effects of excess iron.

Translational control: the ferritin story

Ferritin is a protein found in all cells of animals and plants and also in bacteria and molds. Ferritin stores iron within its protein shell, and thus provides protection against the accumulation of excessive levels of iron in the cytosol, where the iron can peroxidize unsaturated lipids and damage other cell constituents. Channels penetrating the protein wall of the ferritin molecule allow cytoplasmic iron to enter it and stored iron to be returned to the cytosol for use in essential metabolic processes. At sites not yet clearly defined, as ferrous iron enters it is oxidized to ferric iron for storage as ferric oxyhydroxide.

Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates.

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).

Fortification of curry powder with NaFe(111)EDTA in an iron-deficient population: report of a controlled iron-fortification trial

A targeted, double-blind controlled iron fortification trial using Fe(111)-EDTA in masala (curry powder) was directed towards an Fe-deficient Indian population for 2 y. The Fe status of the fortified group improved more than that of control subjects. Improvement reached significance over control subjects for females in hemoglobin (p = 0.0005), ferritin (p = 0.0002), and body Fe stores (p = 0.001) and for males in ferritin (p = 0.04). The prevalence of Fe-deficiency anemia (IDA) decreased from 22 to 5% in fortified females. Premenopausal women, multipara women, and women with prolonged menstruation or initial IDA benefitted most from fortification. The mean increase in body Fe stores in females with initial IDA was 9.0 +/- 1.3 mmol, representing an increased absorption of 12 mumol/d. Fortified subjects with normal Fe status did not accumulate excessive body Fe and there was no alteration in serum Zn concentrations. Targeted fortification is a safe and effective means of combatting Fe deficiency.

Red cell ferritin and iron stores in patients with chronic disease.

Serum and red cell ferritin were determined in a heterogeneous group of 59 patients with chronic disease undergoing a bone marrow biopsy. There was very little correlation between serum and red cell ferritin (r = 0.53). Although serum ferritin increased in relation to increased bone marrow iron stores, only 1 out of 8 patients with absent marrow iron stores and none of 8 patients with reduced marrow iron stores had a decreased serum ferritin. In contrast, 6 of 8 patients with absent iron stores had a reduced red cell ferritin concentration. There was no significant difference between the mean red cell ferritin of the patients with reduced, normal and mild-moderately increased marrow iron stores (30, 26 and 34 ag/cell). Red cell ferritin was decreased in 78% of a group of 32 patients with a low mean cell volume. In the patients studied, red cell ferritin was a better indicator of absent iron stores than serum ferritin. However, red cell ferritin did not detect a reduction in the iron status until the marrow iron stores were completely depleted. Apparently, during normal erythropoiesis the primitive erythroblasts continue to take up iron irrespective of the amount of iron available in the stores.

Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.

The non-immune inflammatory response: Serial changes in plasma iron, iron-binding capacity, lactoferrin, ferritin and C-reactive protein

The interrelationships between various components of the non-immune inflammatory response (white cell count, plasma lactoferrin, C-reactive protein, ferritin, iron and iron-binding capacity), were studied serially in a variety of inflammatory conditions including acute lobar pneumonia, active pulmonary tuberculosis, rheumatoid arthritis on gold therapy and sepsis in the face of marrow hypoplasia induced by chemotherapy. Lactoferrin concentrations paralleled the white count in all groups. They were highest in pneumonia and tuberculosis, mildly elevated in rheumatoid arthritis and markedly decreased in neutropenic sepsis. Very high initial lactoferrin concentrations were associated with a poor prognosis in acute pneumonia. C-reactive protein and ferritin concentrations remained elevated through the period of study in acute pneumonia and neutropenic sepsis, while they gradually normalised over weeks in subjects with tuberculosis or rheumatoid arthritis on therapy. In pneumonia and tuberculosis moderate hypoferraemia and a reduced iron-binding capacity were evident. In contrast, a raised percentage saturation was present in neutropenic sepsis, probably related to erythroid marrow suppression. Comparisons between ferritin, lactoferrin and C-reactive protein in the various groups supported the concept that ferritin behaves in part as an acute phase reactant and that hypoferraemia in inflammation is due to deviation of iron into ferritin stores. The suggestion that lactoferrin is responsible for the hypoferraemia and hyperferritinaemia was not supported by the present data. Iron deficiency appeared to limit the hyperferritinaemic response in rheumatoid arthritis, while erythropoietic inhibition by chemotherapy dampened the hypoferraemic response in neutropenic sepsis.

Iron storage in ferritin following intracellular hemoglobin denaturation in erythroleukemic cells

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 57Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mossbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-57Fe to ferritin-57Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.

Iron Storage in Ferritin Following Intracellular Hemoglobin Denaturation in Erythroleukemic Cells

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 57Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mössbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-57Fe to ferritin-57Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.

Letter to the Editor: Estimation of Body Iron Stores from Serum Ferritin Concentration

Three methods have thus far been developed to convert a serum ferritin value to the absolute amount of storage iron. Walters et al. (4) noted in 1973 that 1 ng/ml of serum ferritin was equivalent to 8 mg of iron stores. Cook et al. (2) found a similar correlation where 1 ng/ml equalled 10 mg of storage iron. Saarinen and Siimes discovered a linear correlation between the logarithmic value of serum ferritin and the iron stores expressed as mg/kg body weight (3). Table 1 shows the differences between these methods in converting the average serum ferritin values in adult male and female, in children weighing 15 and 30 kg, as well as in the newborn. Strikingly, the methods differ from each other, particularly in the newborn where the methods of Walters et al. and Cook et al. result in marked overestimation: about 30 times as high values as estimated by chemical analyses (5). We agree with Caballero et al. (1) that the estimation in the preschool child is the lowest by the Saarinen and Siimes method. Caballero et al. state that a child weighing 14 kg may have 468 mg of storage iron. This value seems to be unusually high compared to those that are obtained by any other methods. Further, it is unfortunate that they do not give data on the serum ferritin concentration in that particular child because it would have been the first time when a direct comparison between serum ferritin and iron stores has been given. It would also be the real way to compare these three methods. We are confident that none of the three methods fit well all circumstances and all age-groups because critical experiments are

Human ferritin gene is assigned to chromosome 19.

Ferritin is the intracellular iron storage protein. Tissue ferritin stores are markedly increased in hemochromatosis, a disease of iron overload that has been linked to chromosome 6. In order to provide further information concerning the genetics of ferritin synthesis and to determine if the structural gene for ferritin was on chromosome 6, studies were performed to identify the human chromosome that contains the ferritin gene. Ferritin immunoassays were performed on extracts of Chinese hamster ovary somatic cells that were hybridized with human lymphocytes and fibroblasts and contained various human chromosomes in different combinations. None of the 13 cell lines that lacked immunoreactive human ferritin contained chromosome 19, and all 9 of the cell lines that produced human ferritin contained chromosome 19. No other human chromosome shared this association with human ferritin. In studies of subclones of ferritin-positive cell lines, immunoreactive ferritin consistently segregated only with chromosome 19. Immunoprecipitation studies performed on cells that had been incubated with 59Fe-containing transferrin indicated that chromosome 19-containing cells incorporated iron into intact and functional molecules of human ferritin. The necessary and exclusive association of chromosome 19 with human ferritin indicates that a defect in the structural gene for human ferritin cannot account for the abnormalities of hemochromatosis. Moreover, this hamster-human hybrid cell system should prove useful in further studies of regulation of ferritin concentration and composition.

Liver xanthine dehydrogenase and iron mobilization

Administration of a tungsten-supplemented diet to young rats for 10 weeks resulted in an 80% – 90% reduction in the liver xanthine dehydrogenase activity and a 100% increase in the liver non-heme iron content as compared to control animals. A time course study of the effect of dietary tungsten in adult rats demonstrated that an increase in the non-heme iron content of liver was not observed until the liver xanthine dehydrogenase activity was reduced by 50% of that of control animals. Reduction of the liver xanthine dehydrogenase activity beyond 50% of the control value was accompanied by a progressive and continual increase in the non-heme iron content of liver. Removal of the tungsten from the diet of the remaining rats resulted in a rapid increase of the liver xanthine dehydrogenase activity which was accompanied by a decrease in the liver non-heme iron content. These results would support the hypothesis that liver xanthine dehydrogenase participates in the liberation of iron from liver ferritin stores.

Binding of serum ferritin to concanavalin A: patients with homozygous beta thalassaemia and transfusional iron overload.

Serum ferritin concentrations have been measured in 124 patients with homozygous beta thalassaemia who were between 2 and 21 years old, had received 11--504 units of blood but had not undergone splenectomy. There were highly significant correlations between serum ferritin concentration and both the amount of blood transfused and alanine amino-transferase (ALT) activity. However, multivariate analysis showed that units of blood and ALT activity together only accounted for about 30% of the variation in serum ferritin concentration. Little of the remaining variation could be explained by other variables related to iron metabolism or liver damage. The concentration of concanavalin A binding ferritin increased rapidly with the number of units of blood up to 100 units but thereafter showed no further increase with number of transfusions. The concentration of non-binding ferritin was more closely related to transfusion load. These results suggest that the secretion of glycosylated ferritin from reticuloendothelial cells reaches a maximum with increasing iron accumulation, perhaps reflecting a maximum rate of synthesis. Ferritinaemia in patients with transfusional iron overload therefore seems to be the result of the combined effects of increased ferritin synthesis and the release of intracellular ferritin from damaged cells. A simple relationship between serum ferritin and iron stores cannot be assumed when ferritin concentrations exceed 4000 microgram/l or in patients who have received more than 100 units of transfused blood.

Serum ferritin and bone marrow biopsy iron stores. II. Correlation with low serum iron and Fe/TIBC ratio less than 15%.

Diseases associated with defective reticuloendothelial release of iron may be difficult to distinguish from the iron-deficient state, since serum iron parameters (serum iron, percentage saturation of transferrin less then 15%) may overlap. Assessment of the bone marrow biopsy iron stores was often necessary to resolve the diagnosis. Serum ferritin levels have proved very useful in distinguishing the uncomplicated iron-deficient state from other disorders associated with low serum iron and low percentage saturation of transferrin. However, a small percentage of iron-deficient persons may have normal ferritin values, particularly if these persons have liver disease or hematopoietic or lymphoreticular neoplasms. In these cases, assessment of the bone marrow biopsy iron would still be necessary to evaluate the body iron stores.

The clinical significance of ferritinuria

Urinary ferritin levels were measured by a “2-site” immunoradiometric assay in normal volunteers and in patients with various hematologic disorders. The mean urinary ferritin concentration in normal subjects averaged 2.2 microgram/liter, only 3% of the serum ferritin level. Elevated urinary ferritin levels averaging 45 microgram/liter were observed in patients with hematologic malignancies, but there was a proportional increase in serum ferritin so that the urinary level still averaged only 7% of the serum value. The highest urinary ferritin values (mean 170 microgram/liter) were associated with chronic hemolytic anemia, and in these patients, urinary ferritin rose disproportionately in relation to the serum, averaging 82% of it. This higher urinary level apparently reflects increased ferritin in renal tubular cells due to glomerular filtration of unbound hemoglobin, a mechanism that is supported by a highly significant correlation between urinary ferritin and serum haptoglobin levels. In normal subjects and in patients with malignancy, the source of urinary ferritin appears different, since a highly significant correlation was observed between urinary ferritin and reticuloendothelial iron stores as measured by serum ferritin or total iron-binding capacity. In this setting, the most likely source of urinary ferritin is the iron contained in renal tubular cells, which is apparently in equilibrium with body iron stores.

The Clinical Significance of Ferritinuria

Urinary ferritin levels were measured by a "2-site" immunoradiometric assay in normal volunteers and in patients with various hematologic disorders. The mean urinary ferritin concentration in normal subjects averaged 2.2 microgram/liter, only 3% of the serum ferritin level. Elevated urinary ferritin levels averaging 45 microgram/liter were observed in patients with hematologic malignancies, but there was a proportional increase in serum ferritin so that the urinary level still averaged only 7% of the serum value. The highest urinary ferritin values (mean 170 microgram/liter) were associated with chronic hemolytic anemia, and in these patients, urinary ferritin rose disproportionately in relation to the serum, averaging 82% of it. This higher urinary level apparently reflects increased ferritin in renal tubular cells due to glomerular filtration of unbound hemoglobin, a mechanism that is supported by a highly significant correlation between urinary ferritin and serum haptoglobin levels. In normal subjects and in patients with malignancy, the source of urinary ferritin appears different, since a highly significant correlation was observed between urinary ferritin and reticuloendothelial iron stores as measured by serum ferritin or total iron-binding capacity. In this setting, the most likely source of urinary ferritin is the iron contained in renal tubular cells, which is apparently in equilibrium with body iron stores.

Bone marrow iron stores and serum ferritin

Ferritin is an intracellular iron storage protein found mainly in the cytosol of the cells of the reticuloendothelial system. The introduction of sensitive immunoradiometric methods for detecting ferritin has made possible its quantitation in the serum. Serum ferritin is known to correlate well with body iron stores in healthy subjects and subjects with iron deficiency and iron overload. Serum ferritin levels inappropriate to the amount of body iron stores have been reported in subjects with liver disease, malignancy and many other chronic disorders. Total body iron stores are traditionally assessed by cytochemical examination of the bone marrow. However, this method may not be convenient and is sometimes refused by the patient. In contrast serum ferritin is simple, sensitive and reliable. In our present study, we have determined concomitantly the serum ferritin and bone marrow iron stores in a mixed hospital population. The results suggest a correlation between serum ferritin and the bone marrow iron stores. The possibility of using serum ferritin to predict the status of body stores in a mixed hospital population will be discussed.