Mechanisms Of Iron Regulation Research Articles

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

Regulation of iron homeostasis by the p53-ISCU pathway.

Accumulation of iron in tissues increases the risk of cancer, but iron regulatory mechanisms in cancer tissues are largely unknown. Here, we report that p53 regulates iron metabolism through the transcriptional regulation of ISCU (iron-sulfur cluster assembly enzyme), which encodes a scaffold protein that plays a critical role in Fe-S cluster biogenesis. p53 activation induced ISCU expression through binding to an intronic p53-binding site. Knockdown of ISCU enhanced the binding of iron regulatory protein 1 (IRP1), a cytosolic Fe-S protein, to an iron-responsive element in the 5′ UTR of ferritin heavy polypeptide 1 (FTH1) mRNA and subsequently reduced the translation of FTH1, a major iron storage protein. In addition, in response to DNA damage, p53 induced FTH1 and suppressed transferrin receptor, which regulates iron entry into cells. HCT116 p53+/+ cells were resistant to iron accumulation, but HCT116 p53−/− cells accumulated intracellular iron after DNA damage. Moreover, excess dietary iron caused significant elevation of serum iron levels in p53−/− mice. ISCU expression was decreased in the majority of human liver cancer tissues, and its reduced expression was significantly associated with p53 mutation. Our finding revealed a novel role of the p53-ISCU pathway in the maintenance of iron homeostasis in hepatocellular carcinogenesis.

The utility of iron chelators in the management of inflammatory disorders.

Since iron can contribute to detrimental radical generating processes through the Fenton and Haber-Weiss reactions, it seems to be a reasonable approach to modulate iron-related pathways in inflammation. In the human organism a counterregulatory reduction in iron availability is observed during inflammatory diseases. Under pathological conditions with reduced or increased baseline iron levels different consequences regarding protection or susceptibility to inflammation have to be considered. Given the role of iron in development of inflammatory diseases, pharmaceutical agents targeting this pathway promise to improve the clinical outcome. The objective of this review is to highlight the mechanisms of iron regulation and iron chelation, and to demonstrate the potential impact of this strategy in the management of several acute and chronic inflammatory diseases, including cancer.

Iron and Parasites

Iron and Parasites

A High Serum Iron Level Causes Mouse Retinal Iron Accumulation Despite an Intact Blood-Retinal Barrier

The retina can be shielded by the blood-retinal barrier. Because photoreceptors are damaged by excess iron, it is important to understand whether the blood-retinal barrier protects against high serum iron levels. Bone morphogenic protein 6 (Bmp6) knockout mice have serum iron overload. Herein, we tested whether the previously documented retinal iron accumulation in Bmp6 knockout mice might result from the high serum iron levels or, alternatively, low levels of retinal hepcidin, an iron regulatory hormone whose transcription can be up-regulated by Bmp6. Furthermore, to determine whether increases in serum iron can elevate retinal iron levels, we i.v. injected iron into wild-type mice. Retinas were analyzed by real-time quantitative PCR and immunofluorescence to assess the levels of iron-regulated genes/proteins and oxidative stress. Retinal hepcidin mRNA levels in Bmp6 knockout retinas were the same as, or greater than, those in age-matched wild-type retinas, indicating that Bmp6 knockout does not cause retinal hepcidin deficiency. Changes in mRNA levels of L ferritin and transferrin receptor indicated increased retinal iron levels in i.v. iron-injected wild-type mice. Oxidative stress markers were elevated in photoreceptors of mice receiving i.v. iron. These findings suggest that elevated serum iron levels can overwhelm local retinal iron regulatory mechanisms.

α-Actinin TvACTN3 ofTrichomonas vaginalisIs an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

Inflammatory regulation of iron metabolism during thioacetamide-induced acute liver injury in rats

Systemic iron homeostasis is tightly regulated by the interaction between iron regulatory molecules, mainly produced by the liver. However, the molecular mechanisms of iron regulation in liver diseases remain to be elucidated. Here we analyzed the expression profiles of iron regulatory molecules during transient iron overload in a rat model of thioacetamide (TAA)-induced acute liver injury. After TAA treatment, mild hepatocellular degeneration and extensive necrosis were observed in the centrilobular region at hour 10 and on day 1, respectively. Serum iron increased transiently at hour 10 and on day 1, in contrast to hypoferremia in other rodent models of acute inflammation reported previously. Thereafter, up-regulation of hepcidin, a central regulator of systemic iron homeostasis, was observed in hepatocytes on day 2. Expression of transferrin receptor 1 and ferritin subunits increased to a peak on day 3, followed by increases in liver iron content and stainable iron on day 5, in parallel with regeneration of hepatocytes. Histopathological lesions and hepatocellular iron accumulation disappeared until day 10. The hepcidin induction was preceded by activation of IL6/STAT3 pathway, whereas other pathways known to induce hepcidin were down-regulated. IL6 was expressed by MHC class II-positive macrophages in the portal area, suggestive of dendritic cells. Our results suggest that IL6 released by portal macrophages may regulate hepatocyte hepcidin expression via STAT3 activation during transient iron overload in TAA-induced acute liver injury.

Impact of diet and weight loss on iron and zinc status in overweight and obese young women.

Young overweight women are at risk of iron and zinc deficiency. This study assessed iron, zinc and inflammatory status during a 12-month weight loss trial in young women (18-25 y; BMI >=27.5 kg/m2) randomised to a higher-protein (HP: 32% protein; 12.2 mg/day iron; 11.7 mg/day zinc) or lower-protein (LP: 20%; 9.9 mg/day; 7.6 mg/day respectively) diet with contrasting haem iron and zinc content. In completers (HP: n=21; LP: n=15), HP participants showed higher median ferritin (52.0 vs 39.0 μg/L; p=0.021) and lower median soluble transferrin receptor-ferritin index (sTfR-F; 0.89 vs 1.05; p=0.024) although concentrations remained within normal range for both diets. Median C-reactive protein (CRP; HP: 3.54; LP: 4.63 mg/L) and hepcidin (HP: 5.70; LP: 8.25 ng/mL) were not elevated at baseline, and no longitudinal between-diet differences were observed for zinc and CRP. Compared to those with <5% weight loss, HP participants losing >=10% weight showed lower median sTfR-F (0.76 vs 1.03; p=0.019) at six months. Impact of >=10% weight loss on iron was more apparent in LP participants who exhibited greater mean serum iron (20.0 vs 13.5 μmol/L; p=0.002), transferrin saturation (29.8% vs 19.4%; p=0.001) and lower sTfR (1.24 vs 1.92 mg/L; p=0.034) at 12 months. Results show normal iron and zinc status can be maintained during 12 months of energy restriction. In the absence of elevated baseline inflammation and hepcidin, a more favourable iron profile in those with >=10% weight loss may reflect stronger compliance or the potential influence of iron regulatory mechanisms unrelated to inflammatory hepcidin reduction.

Brain Iron Homeostasis: From Molecular Mechanisms To Clinical Significance and Therapeutic Opportunities

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

Deferiprone for the treatment of Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurological disease related to a deficiency of the protein frataxin involved in iron-sulfur (Fe-S) cluster biogenesis. This leads to an increased cellular iron uptake accumulating in mitochondria, and a subsequently disturbed iron homeostasis. The detailed mechanism of iron regulation of frataxin expression is yet unknown. Deferiprone, an iron chelator that may cross the blood-brain barrier, was shown to shuttle iron between subcellular compartments. It could also transfer iron from iron-overloaded cells to extracellular apotransferrin and pre-erythroid cells for heme synthesis. Here, clinical studies on Deferiprone are reviewed in the context of alternative agents such as desferoxamine, with specific regard to its mechanistic and clinical implications.

Virulence mechanisms of bacterial aquaculture pathogens and antivirulence therapy for aquaculture

AbstractAlthough aquaculture continues to be the fastest‐growing food‐producing industry worldwide, the sector faces important challenges with respect to controlling infectious diseases. Indeed, the frequent use of antibiotics, which are traditionally the most important line of defence against bacterial disease, has raised problems related to the development and spread of resistance. As a result, we currently are in a race to develop novel therapeutics that should allow us to continue controlling bacterial disease in the future. In this respect, scientific progress of the past years has allowed us to start understanding the mechanisms by which bacterial pathogens are causing disease. This knowledge in its turn opens up novel possibilities to combat infections by interfering with these mechanisms, a strategy that has been termed antivirulence therapy. In this review, the current knowledge with respect to pathogenicity mechanisms of bacterial aquaculture pathogens is discussed, including adhesion mechanisms, production of extracellular polysaccharides and biofilm formation, production of lytic enzymes (e.g. haemolysins and proteases), mechanisms of iron acquisition, secretion systems and regulatory mechanisms such as quorum sensing and host–pathogen signalling with catecholamine stress hormones. Furthermore, an overview is given of the first steps towards antivirulence therapy for aquaculture, with a focus on quorum sensing disruption as a proof of concept.

Decreased iron levels in the temporal cortex in postmortem human brains with Parkinson disease

The present study aimed to evaluate alterations in the levels of iron, divalent metal transporter 1 (DMT1) with the iron-responsive element (IRE), transferrin receptor 1 (TfR1), ferroportin 1 (FPN1), and iron regulatory protein 1 (IRP1) in the temporal cortex of human brains with Parkinson disease (PD). Iron content was measured using an ICP-MS 7500CE detector. IRP1, DMT1+IRE, TfR1, and FPN1 expressions were determined by Western blotting. Iron content was significantly lower in the temporal cortex of patients with PD when compared with age-matched healthy controls. Unexpectedly, the levels of DMT1+IRE, TfR1, FPN1, and IRP1 were decreased in the temporal cortex in PD brains. No changes were observed in the temporal cortex of postmortem Alzheimer disease brains. Iron deprivation and iron-related protein dysregulation suggest that a different iron regulatory mechanism may exist, and that iron redistribution may occur between the temporal cortex and the substantia nigra of patients with PD.

Effects of hemochromatosis and transferrin gene mutations on peripheral iron dyshomeostasis in mild cognitive impairment and Alzheimer's and Parkinson's diseases

Deregulation of iron metabolism has been observed in patients with neurodegenerative diseases. We have carried out a molecular analysis investigating the interaction between iron specific gene variants [transferrin (TF, P589S), hemochromatosis (HFE) C282Y and (H63D)], iron biochemical variables [iron, Tf, ceruloplasmin (Cp), Cp:Tf ratio and % of Tf saturation (% Tf-sat)] and apolipoprotein E (APOE) gene variants in 139 Alzheimer's disease (AD), 27 Mild Cognitive Impairment (MCI), 78 Parkinson's disease (PD) patients and 139 healthy controls to investigate mechanisms of iron regulation or toxicity. No difference in genetic variant distributions between patients and controls was found in our Italian sample, but the stratification for the APOEε4 allele revealed that among the APOEε4 carriers was higher the frequency of those carriers of at least a mutated TF P589S allele. Decreased Tf in both AD and MCI and increased Cp:Tf ratio in AD vs. controls were detected. A multinomial logistic regression model revealed that increased iron and Cp:Tf ratio and being man instead of woman increased the risk of having PD, that increased values of Cp:Tf ratio corresponded to a 4-fold increase of the relative risk of having MCI, while higher Cp levels were protective for PD and MCI. Our study has some limitations: the small size of the samples, one ethnic group considered, the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest the hypothesis that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk.

NGP1-01, a multi-targeted polycyclic cage amine, attenuates brain endothelial cell death in iron overload conditions

Development and progression of neurodegenerative disorders have, amongst other potential causes, been attributed to a disruption of iron regulatory mechanisms and iron accumulation. Excess extracellular iron may enter cells via nontraditional routes such as voltage-gated calcium channels and N-methyl-d-aspartate (NMDA) receptors leading to intracellular oxidative damage and ultimately mitochondrial failure. Nimodipine, an L-type calcium channel blocker has been shown to reduce iron-induced toxicity in neuronal and brain endothelial cells. Our current study investigates NGP1-01, a multimodal drug acting as an antagonist at both the NMDA receptor and the L-type calcium channel. Our previous studies support NGP1-01 as a promising neuroprotective agent in diseases involving calcium-related excitotoxicity. We demonstrate here that NGP1-01 (1 and 10μM) pretreatment abrogates the effects of iron overload in brain endothelial cells protecting cellular viability. Both concentrations of NGP1-01 were found to attenuate iron-induced reduction in cellular viability to a similar extent, and were statistically significant. To further verify the mechanism, the L-type calcium channel agonist FPL 64176 was administered to promote iron uptake. Addition of NGP1-01 dose-dependently reduced FPL 64176 stimulated uptake of iron. These data support further evaluation of NGP1-01 as a neuroprotective agent, not only in diseases associated with excitotoxicity, but also in those of iron overload.

Targeting the hepcidin–ferroportin axis to develop new treatment strategies for anemia of chronic disease and anemia of inflammation

Anemia of chronic disease (ACD) or anemia of inflammation is prevalent in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease. ACD is associated with poor prognosis and lower quality of life. Management of ACD using intravenous iron and erythropoiesis stimulating agents are ineffective for some patients and are not without adverse effects, driving the need for new alternative therapies. Recent advances in our understanding of the molecular mechanisms of iron regulation reveal that increased hepcidin, the iron regulatory hormone, is a key factor in the development of ACD. In this review, we will summarize the role of hepcidin in iron homeostasis, its contribution to the pathophysiology of ACD, and novel strategies that modulate hepcidin and its target ferroportin for the treatment of ACD.

Regulation of Flvcr by Macrophages in Response to Lipopolysaccharide

Abstract 2111Pathogenic bacteria must acquire iron from their hosts to survive and have evolved multiple mechanisms to capture iron or iron-containing heme from the bloodstream or tissues. In response, mammals have developed defense mechanisms to keep iron from pathogens. For example, in response to inflammatory cytokines, hepcidin secreted by the liver binds to the iron exporter ferroportin (FPN1), leading to FPN1 internalization and degradation, decreasing gastrointestinal iron absorption and increasing macrophage iron storage. Much of the body's iron stores are complexed in heme. The Feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, is a heme export protein. We showed previously that FLVCR is required for the normal development of the erythroid [Science (2008)319:825] and T cell lineages [Blood (ASH Annual Meeting Abstracts)114:913,2009].Although macrophages express high levels of FLVCR, the role of FLVCR in regulating heme-iron after infection remains unexplored. Other heme regulatory proteins, such as heme oxygenase-1 (HMOX1), a heme-degrading enzyme, are known to be transcriptionally regulated in macrophages in response to infection. We hypothesized that macrophages dynamically regulate Flvcr in response to bacterial infection. To test this hypothesis, we stimulated J774, a murine macrophage cell line, with lipopolysaccharide (LPS from E. coli O111:B4) at varying concentrations and durations. LPS, an outer membrane component from gram-negative bacteria, binds to Toll-like receptor 4 (TLR4) on macrophages and activates downstream signaling pathways. Using multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR), we measured mRNA levels of Flvcr, Hmox1, and Fpn1. We found that J774 cells down-regulated Flvcr transcript levels in response to LPS with a maximal decrease (69%) seen at 6–8 hours of stimulation. While the extent of Flvcr down-regulation was dose-responsive, a significant decrease (57%) occurred even with the lowest LPS dose (10 ng/ml). Macrophages decreased Fpn1 expression (71%) and increased Hmox1 expression (55%) in response to LPS stimulation as previously reported. Similar results were obtained with LPS from a different bacterial source (Salmonella minnesota Re595). We also performed these studies using primary macrophages cultured from murine bone marrow mononuclear cells and observed a similar decrease in Flvcr and Fpn1 (64 and 72%) and an increase in Hmox1 (40%) transcripts after stimulation with both O111:B4 and Re595 LPS.While Fpn1 transcriptional regulation by heme and oxidative stress has been studied, the mechanism by which LPS regulates Fpn1 transcription is less clear. The similar pattern and kinetics of LPS-induced Flvcr and Fpn1 expression changes raise the possibility that the same regulatory mechanism is responsible. Analysis of the human and mouse Flvcr promoter regions revealed several putative LPS downstream transcription factor binding sites including NF-κB, AP1, and C/EBPβ. In addition to transcriptional regulation, LPS downstream signaling could alter Flvcr and Fpn1 mRNA stability and translation, so we compared the 5’ untranslated regions (UTR) and 3’UTR of murine Flvcr and Fpn1. We found little similarity between the 5’UTR of Flvcr and the 5’UTR of Fpn1, known to contain an iron-responsive element (IRE) and be regulated by iron via iron regulatory proteins (IRP). However, alignment of the 3’UTR from Flvcr and Fpn1 showed similarity (pair wise score 65). Both the Flvcr and Fpn1 3’UTR are predicted to have a high degree of secondary structure based on their large negative fold energies (−421.25 and −300.74 kcal/mol), further suggesting that these 3’UTR may have a regulatory function. Studies are underway to determine the roles of the Flvcr promoter, 5’UTR, and 3’UTR in LPS-induced down-regulation.This work suggests that LPS-induced down-regulation of Flvcr and Fpn1 might act in concert to decrease heme and iron export from macrophages and sequester iron from bacterial pathogens. Heme export control through FLVCR could serve as a novel mechanism of iron regulation in response to infection. Disclosures:No relevant conflicts of interest to declare.

Hepcidin in Male Double Red Blood Cell Donors - Relationship Between Parameters of Iron Metabolism and Erythropoiesis

Abstract 2109Hemoglobin (Hb) below 12.5g/dL accounts for more than 40% of all blood donor deferrals, and no standardized message is provided to these deferred donors. Furthermore, many donors who meet Hb standards are iron depleted. In light of this, the FDA requested comments on possibly changing the minimal acceptable Hb and/or the inter-donation interval. Here we explore several parameters of iron utilization after red blood cell (RBC) donation to understand its relationship to erythropoiesis.Hepcidin is the central regulator of iron absorption, suppressed by low iron stores. We hypothesize that hepcidin values would identify donors with normal Hb levels and low iron stores and thus identify those who would benefit from iron replacement to prevent future low Hb deferral.We enrolled eligible repeat male double RBC donors randomized to receive placebo (n=5) or iron supplementation (n=5) using 90 mg of elemental iron (feosol caplets). Donors were enrolled on day of donation and were invited to return at 2–4 day intervals during the 14 days following donation to compare with pre-donation markers of erythropoiesis and iron metabolism. In addition, buffy coats were collected on day 0 and again 7–10 days following donation (n=6) to quantify by flow cytometry circulating CD34+ cells as well as numbers of common myeloid progenitors (CMPs) and megakaryocyte erythroid progenitors (MEPs) in study donors.Comparing iron supplemented to placebo receiving volunteers, ferritin was higher at 14 days in iron supplemented donors (18.6±3.9 vs 12.0±1.6 ng/mL) and corresponds with 52% vs. 36% of levels prior to donation relative to placebo (P=0.05). Furthermore, serum hepcidin nadirs earlier and rebounds sooner relative to placebo (15.4±3.2 vs. 6.8±2.1 ng/mL, P=0.05). Serum iron (150±37.1 vs. 60±5.8 mg/dL, P=0.04) and transferrin saturation (39.8±9.8 vs. 16.2±2.1%, P=0.05) are also increased relative to placebo by day 10 following donation. Although we found no difference in recovery of RBC count, hemoglobin, or hematocrit following donation, a trend toward a more sustained elevation in reticulocyte count is present relative to placebo (158±12.1 vs. 118±17.3 × 109 cells/mL, P=0.07). CD34+ cells in circulation are increased following donation in all donors. Furthermore, only donors with baseline ferritin levels >20 ng/mL increased MEPs following donation. These later findings are consistent with expectations of iron regulatory mechanisms influencing erythropoiesis.Taken together, these data suggest that a complex interplay between iron regulatory and erythropoietic mechanisms occurs during recovery from blood donation. Although it is currently unclear how iron regulation affects erythropoiesis, a larger (double) sample size will aid in the generation of criteria for donor iron supplementation. Disclosures:Olbina:Intrinsic LifeSciences LLC: Employment, Equity Ownership; Hepcidin C-ELISA:. Westerman:Intrinsic LifeSciences LLC: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Hepcidin C-ELISA: Patents & Royalties; Centocor-Ortho R&D, Inc.: Honoraria.

Structural and functional analyses of chicken liver ferritin

Characterization of ferritins from different species has provided insight into iron regulation mechanisms and evolutionary relationships. Here, we examined chicken liver ferritin, which comprises only H subunit and has 14.8 µg of Fe/100 µg of protein. The chicken H subunit apo homopolymer showed the same iron uptake rate as bovine H subunit homopolymer expressed with a baculovirus expression system (0.31 and 0.28 mmol of Fe/min per micromole of protein for chicken and bovine H subunit, respectively). Chicken H subunit apo homopolymer showed a significantly higher biotinylated hemin-binding activity than liver holoferritin. Although bovine spleen apoferritin, which has an L (liver or light):H (heart or heavy) subunit ratio of 1:1, also shows a significantly higher biotinylated hemin-binding activity than its holoferritin, these biotinylated hemin-binding activities were markedly lower than those of both chicken holo- and apoferritins. Binding of chicken holo- and apoferritin with biotinylated hemin was strongly inhibited by hemin but not iron-free hemin, protoporphyrin IX, or Zn-protoporphyrin. These findings demonstrate that chicken ferritin comprises only an H subunit, possesses ferroxidase activity as in mammalian ferritin H subunits, and binds heme more strongly than mammalian ferritins.

The effect of age on intestinal iron uptake

Background: The pro-oxidant role of iron in neurodegenerative and chronic disease has led to concern over the danger of excessive levels posing a health risk (Kell, 2009). As the body has limited capacity to excrete iron, absorption acts as the main homeostatic mechanism (Anderson et al., 2009). Although iron stores increase with age (Liu et al., 2003), the effect of advancing age on iron regulation has not been extensively studied and age related misregulation has been implied (Levenson et al., 2004). The aim of this study was to investigate the effect of age on intestinal iron uptake in adults. Method: Thirty-five participants (29 females and six males subdivided into three age groups) provided fasted blood samples. A second sample was taken three hours following a standard breakfast (low in iron inhibitors and enhancers) and a 200 mg iron sulphate supplement as used by Hutchinson et al. (2004). Total plasma iron, total iron binding capacity (TIBC), transferrin saturation (TSAT) and non-transferrin bound iron (NTBI) were measured. Participants completed a 52 item food frequency questionnaire, adapted from the validated EPIC-Norfolk study to be iron specific. Data was statistically analysed using anova and Pearson regression. Approval was granted by the University of Plymouth Ethical Committee. Results: Total plasma iron absorption and post supplement increases in TSAT and NTBI were greatest in the 35–55 years age group (Table 1). TIBC and iron intake were similar for all groups. A weak correlation was found between iron uptake and change in NTBI (r = 0.405, P < 0.022). Table 1. Mean (SD) plasma analysis, standard deviation and significance between groups Variable 18–34 years 35–55 years 56+ years Significance Δ = change, PP = postprandial, TIBC = total iron binding capacity, TSAT = transferrin saturation. Δ In Iron μM 11.9 (17.4) 20.8 (10) 6.2 (7.4) P < 0.051 Δ In NTBI μM 0.24 (0.36) 1.09 (1.48) 0.29 (0.5) P < 0.060 TIBC μM 102 (23) 92 (23) 97 (11) P < 0.525 % TSAT (PP) 49 (13) 60 (9) 41 (9) P < 0.014 Iron Intake (mg) 66 (29) 67 (21) 73 (1) P < 0.747 Discussion: This study does not support the suggestion that iron absorption is misregulated in older age (Levenson et al., 2004) but does support the ‘set point’ theory that iron uptake increases with age until a certain point (Garry et al., 2000). In healthy individuals the plasma concentration of NTBI is small; the elevated amount found in the 35–55 year age group is of some concern. This study examined a small, predominantly female sample and the results may have been affected by factors that were not considered such as menstruation, menopause and contraceptive use. Conclusion: It is possible that iron absorption is greater in the middle years of life and that this increases both stores and pro-oxidant damage, which in later years manifests into chronic disease. This study may provide new insight into iron regulation and warrants further research.

Mechanisms of hepatic iron accumulation with aging and environmental heat stress

Regulation of physiological iron stores is crucial for maintenance of homeostasis. Hepatic iron concentrations increase with aging and physiological challenges, which may result in oxidative injury after a stressor. The purpose of this study was to investigate mechanisms of iron regulation in these contexts. The iron regulatory hormone, hepcidin, is induced in response to increases in hepatic iron stores and inflammation; thus, we hypothesized that both aging and hyperthermia would be associated with elevated hepcidin. Young (6 mo) and old (24 mo) Fischer 344 rats were exposed to two bouts of environmental heat stress, and liver and blood samples were taken after the second heating. Hepatic hepcidin expression was evaluated using qPCR, and plasma iron was measured. Because hepcidin is induced by IL-6, and heat stress is an inflammatory challenge, the plasma concentration of IL-6 was also assayed. Heat stress was associated with significant hypoferremia in both age groups two hours after hyperthermia. IL-6 was elevated in old compared to young rats, and was induced by heat stress in both age groups immediately (0 h) after hyperthermia. Hepcidin was induced only in old rats at the two-hour timepoint. These results suggest that hepcidin may have a role in lowering plasma iron after hyperthermia in old rats, but that hypoferremia after heat stress in young rats involves a mechanism distinct from the IL-6 – hepcidin axis.