Scielo RSS <![CDATA[Biological Research]]> vol. 39 num. 1 lang. en <![CDATA[SciELO Logo]]> <![CDATA[Summary of the Third International Workshop on Iron and Copper Homeostasis]]> <![CDATA[Compensatory responses induced by oxidative stress in Alzheimer disease]]> Oxidative stress occurs early in the progression of Alzheimer disease, significantly before the development of the pathologic hallmarks, neurofibrillary tangles and senile plaques. In the first stage of development of the disease, amyloid-β deposition and hyperphosphorylated tau function as compensatory responses and downstream adaptations to ensure that neuronal cells do not succumb to oxidative damage. These findings suggest that Alzheimer disease is associated with a novel balance in oxidant homeostasis. <![CDATA[Molecular and pathological basis of aceruloplasminemia]]> Aceruloplasminemia is an autosomal recessive neurodegenerative disease characterized by iron accumulation in the brain as well as visceral organs. It is a loss-of-function disorder caused by mutations in the ceruloplasmin gene. Clinically, this disease consists of the triad of adult-onset neurological disease, retinal degeneration and diabetes mellitus. Massive iron accumulation and extensive loss of neurons are observed in the basal ganglia. The elevated iron concentration is associated with increased lipid peroxidation in the brains of aceruloplasminemia patients. Enlarged or deformed astrocytes and spheroid-like globular structures are characteristic neuropathological findings in aceruloplasminemia. Moreover, deformed astrocytes and globular structures react positively to anti-4-hydroxynonenal antibody, suggesting that increased oxidative stress is involved in neuronal cell death in aceruloplasminemia brain. More than 30 aceruloplasminemia-causing mutations in the ceruloplasmin gene have been identified. We examined the biosynthesis of two missense ceruloplasmin proteins that result from a Japanese P177R mutation and a Dutch G631R mutation, using Chinese hamster ovary cell expression system. The P177R mutant protein is retained in the endoplasmic reticulum. The G631R mutant protein, predicted to alter the interactions at a single type I copper-binding site, prevented incorporation of copper into apoceruloplasmin and resulted in the synthesis and secretion only of apoceruloplasmin. Molecular analysis of missense mutations showed different structure-function relationships in ceruloplasmin protein. The investigation of mutant ceruloplasmin reveals new insights into molecular pathogenesis of aceruloplasminemia as well as biosynthesis, trafficking, and function of ceruloplasmin. <![CDATA[Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum]]> Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during post-natal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia. <![CDATA[<strong>The functional links between prion protein and copper </strong>]]> Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed. <![CDATA[Homeostatic and toxic mechanisms regulating manganese uptake, retention, and elimination]]> This review attempts to summarize and clarify our basic knowledge as to the various factors that potentially influence the risks imposed from chronic exposure to high atmospheric levels of manganese (Mn). The studies describe the interrelationship of the different systems in the body that regulate Mn homeostasis by characterizing specific, biological components involved in its systemic and cellular uptake and its elimination from the body. A syndrome known as manganism occurs when individuals are exposed chronically to high levels of Mn, consisting of reduced response speed, intellectual deficits, mood changes, and compulsive behaviors in the initial stages of the disorder to more prominent and irreversible extrapyramidal dysfunction resembling Parkinson's disease upon protracted exposure. Mn intoxication is most often associated with occupations in which abnormally high atmospheric concentrations prevail, such as in welding and mining. There are three potentially important routes by which Mn in inspired air can gain access the body to: 1) direct uptake into the CNS via uptake into the olfactory or trigeminal presynaptic nerve endings located in the nasal mucosa and the subsequent retrograde axonal transport directly into the CNS; 2) transport across the pulmonary epithelial lining and its subsequent deposition into lymph or blood; and/or 3) mucocilliary elevator clearance from the lung and the subsequent ingestion of the metal in the gastrointestinal tract. Each of these processes and their overall contribution to the uptake of Mn in the body is discussed in this review as well as a description of the various mechanisms that have been proposed for the transport of Mn across the blood-brain barrier which include both a transferrin-dependent and a transferrin-independent process that may involve store-operated Ca channels. <![CDATA[<strong>Translational control of ceruloplasmin gene expression</strong>: <strong>Beyond the IRE</strong>]]> Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor; erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-γ in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation. <![CDATA[<strong>Iron homeostasis in the lung </strong>]]> Iron is essential for many aspects of cellular function. However, it also can generate oxygen-based free radicals that result in injury to biological molecules. For this reason, iron acquisition and distribution are tightly regulated. Constant exposure to the atmosphere results in significant exposure of the lungs to catalytically active iron. The lungs have a mechanism for detoxification to prevent associated generation of oxidative stress. Those same proteins that participate in iron uptake in the gut are also employed in the lung to transport iron intracellularly and sequester it in an inactive form within ferritin. The release of metal is expedited (as transferrin and ferritin) from lung tissue to the respiratory lining fluid for clearance by the mucocilliary pathway or to the reticuloendothelial system for long-term storage. This pathway is likely to be the major method for the control of oxidative stress presented to the respiratory tract. <![CDATA[<strong>DMT1</strong>: <strong>Which metals does it transport? </strong>]]> DMT1 _ Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 _ transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMT1 also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases ~50x over endogenous expression and ~10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb~Co~Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport. <![CDATA[<strong><i>Cop-like</i></strong><strong> operon</strong>: <strong>Structure and organization in species of the <i>Lactobacillale</i> order</strong>]]> Copper is an essential and toxic trace metal for bacteria and, therefore, must be tightly regulated in the cell. Enterococcus hirae is a broadly studied model for copper homeostasis. The intracellular copper levels in E. hirae are regulated by the cop operon, which is formed by four genes: copA and copB that encode ATPases for influx and efflux of copper, respectively; copZ that encodes a copper chaperone; and copY, a copper responsive repressor. Since the complete genome sequence for E. hirae is not available, it is possible that other genes may encode proteins involved in copper homeostasis. Here, we identified a cop-like operon in nine species of Lactobacillale order with a known genome sequence. All of them always encoded a CopY-like repressor and a copper ATPase. The alignment of the cop-like operon promoter region revealed two CopY binding sites, one of which was conserved in all strains, and the second was only present in species of Streptococcus genus and L. johnsonii. Additional proteins associated to copper metabolism, CutC and Cupredoxin, also were detected. This study allowed for the description of the structure and organization of the cop operon and discussion of a phylogenetic hypothesis based on the differences observed in this operon's organization and its regulation in Lactobacillale order. <![CDATA[Inhibition of iron and copper uptake by iron, copper and zinc]]> Interactions of micronutrients can affect absorption and bioavailability of other nutrients by a number of mechanisms. In aqueous solutions, and at higher uptake levels, competition between elements with similar chemical characteristics and uptake process can take place. The consequences of these interactions may depend on the relative concentrations of the nutrients. In this work, we measure the effects of increasing concentrations of iron, zinc, and copper on iron and copper uptake in Caco-2 cells. Intracellular Fe or Cu levels were affected by incubating with increased concentrations of metals. However, when the cells already had different intracellular metal concentration, the uptake of Fe or Cu was nor affected. In competition studies, we showed that Cu and Zn inhibited Fe uptake, and while Fe inhibited Cu uptake, Zn did not. When the three metals were given together (1: 1: 1 ratio), Fe or Cu uptake was inhibited ~40%. These results point to a potential risk in the absorption and bioavailability of these minerals by the presence of other minerals in the diet. This aspect must be considered in food supplementation and fortification programs. <![CDATA[Antioxidant responses of cortex neurons to iron loading]]> Brain cells have a highly active oxidative metabolism, yet they contain only low to moderate superoxide dismutase and catalase activities. Thus, their antioxidant defenses rely mainly on cellular reduced glutathione levels. In this work, in cortical neurons we characterized viability and changes in reduced and oxidized glutathione levels in response to a protocol of iron accumulation. We found that massive death occurred after 2 days in culture with 10 mM Fe. Surviving cells developed an adaptative response that included increased synthesis of GSH and the maintenance of a glutathione-based reduction potential. These results highlight the fundamental role of glutathione homeostasis in the antioxidant response and provide novel insights into the adaptative mechanisms of neurons subjected to progressive iron loads. <![CDATA[Possible roles of the hereditary hemochromatosis protein, HFE, in regulating cellular iron homeostasis]]> Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein, HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease, Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. This review gives the background and a model as to possible mechanisms of how HFE could exert different effects on iron homeostasis in different cell types. <![CDATA[Hereditary hemochromatosis: An opportunity for gene therapy]]> Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen; iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes <![CDATA[Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines]]> The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways. <![CDATA[<strong>Vesicular transport of Fe and interaction with other metal ions in polarized Caco2 Cell monolayers </strong>]]> Two aspects of the mechanisms by which iron is absorbed by the intestine were studied in the Caco2 cell model, using 59Fe(II)-ascorbate. Data showing the importance of vesicular processes and cycling of apotransferrin (apoTf) to uptake and overall transport of Caco2 cell monolayers (or basolateral 59Fe release) were obtained by comparing effects of: a) adding apoTf to the basal chamber; b) adding vesicular transport inhibitors; or c) cooling to 4ÂșC. These showed that apoTf may be involved in as much as half of Fe transfer across the basolateral membrane, and that vesicular processes may also play a role in non-apoTf-dependent Fe transport. Studies were initiated to examine potential interactions of other metal ions with Fe(II) via DMT1. Kinetic data showed a single, saturable process for uptake of Fe(II) that was pH dependent and had a Km of 7 μM. An excess of Mn(II) and Cu(I) over Fe(II) of 200: 1 (μM: μM) in 1 mM ascorbate markedly inhibited Fe uptake. The kinetics were not competitive. Km increased and Vmax decreased. We conclude that vesicular transport, involving endo- and exocytosis at both ends of the enterocyte, is a fundamental aspect of intestinal iron absorption and that DMT1 may function as a transporter not just for divalent but also for monovalent metal ions. <![CDATA[Iron and glutathione at the crossroad of redox metabolism in neurons]]> Neurons, as non-dividing cells, encounter a myriad of stressful conditions throughout their lifespan. In particular, there is increasing evidence that iron progressively accumulates in the brain with age and that iron-induced oxidative stress is the cause of several forms of neurodegeneration. Here, we review recent evidence that gives support to the following notions: 1) neuronal iron accumulation leads to oxidative stress and cell death; 2) neuronal survival to iron accumulation associates with decreased expression of the iron import transporter DMT1 and increased expression of the efflux transporter IREG1; and 3) the adaptive process of neurons towards iron-induced oxidative stress includes a marked increase in both the expression of the catalytic subunit of gamma glutamate-cysteine ligase and glutathione. These findings may help to understand aging-related neurodegeneration hallmarks: oxidative damage, functional impairment and cell death. <![CDATA[Iron at the center of ferritin, metal/oxygen homeostasis and novel dietary strategies]]> Bioiron _ central to respiration, photosynthesis and DNA synthesis and complicated by radical chemistry with oxygen _ depends on ferritin, the super family of protein nanocages (maxi-ferritins in humans, animals, plants and bacteria, and mini-ferritins, also called DPS proteins, in bacteria) for iron and oxygen control. Regulation of ferritin synthesis, best studied in animals, uses DNA transcription and mRNA translation check points. Ferritin is a member of both the "oxidant stress response" gene family that includes thioredoxin reductase and quinine reductase, and a member of the iron responsive gene family that includes ferroportin and mt-aconitase ferritin DNA regulation responds preferentially to oxidant response inducers and ferritin mRNA to iron inducers; heme confers regulator synergy. Ferritin proteins manage iron and oxygen, with ferroxidase sites and iron + oxygen substrates to form mineral of both Fe and O atoms; maxi-ferritins contribute more to cellular iron metabolism and mini-ferritins to stress responses. Iron recovery from ferritin is controlled by gated protein pores, possibly contributing to iron absorption from ferritin, a significant dietary iron source. Ferritin gene regulation is a model for integrating DNA/mRNA controls, while ferritin protein function is central to molecular nutrition cellular metabolism at the crossroads of iron and oxygen in biology <![CDATA[Divalent cations as modulators of neuronal excitability: Emphasis on copper and zinc]]> Based on indirect evidence, a role for synaptically released copper and zinc as modulators of neuronal activity has been proposed. To test this proposal directly, we studied the effect of copper, zinc, and other divalent cations on voltage-dependent currents in dissociated toad olfactory neurons and on their firing rate induced by small depolarizing currents. Divalent cations in the nanomolar range sped up the activation kinetics and increased the amplitude of the inward sodium current. In the micromolar range, they caused a dose dependent inhibition of the inward Na+ and Ca2+ currents (I Na and I Ca) and reduced de amplitude of the Ca2+-dependent K+ outward current (I Ca-K). On the other hand, the firing rate of olfactory neurons increased when exposed to nanomolar concentration of divalent cations and decreased when exposed to micromolar concentrations. This biphasic effect of divalent cations on neuronal excitability may be explained by the interaction of these ions with high and low affinity sites in voltage-gated channels. Our results support the idea that these ions are normal modulators of neuronal excitability <![CDATA[Understanding copper homeostasis in humans and copper effects on health]]> Based on indirect evidence, a role for synaptically released copper and zinc as modulators of neuronal activity has been proposed. To test this proposal directly, we studied the effect of copper, zinc, and other divalent cations on voltage-dependent currents in dissociated toad olfactory neurons and on their firing rate induced by small depolarizing currents. Divalent cations in the nanomolar range sped up the activation kinetics and increased the amplitude of the inward sodium current. In the micromolar range, they caused a dose dependent inhibition of the inward Na+ and Ca2+ currents (I Na and I Ca) and reduced de amplitude of the Ca2+-dependent K+ outward current (I Ca-K). On the other hand, the firing rate of olfactory neurons increased when exposed to nanomolar concentration of divalent cations and decreased when exposed to micromolar concentrations. This biphasic effect of divalent cations on neuronal excitability may be explained by the interaction of these ions with high and low affinity sites in voltage-gated channels. Our results support the idea that these ions are normal modulators of neuronal excitability <![CDATA[Effect of iron on the activation of the MAPK/ERK pathway in PC12 neuroblastoma cells]]> Recent evidence suggests that reactive oxygen species function as second messenger molecules in normal physiological processes. For example, activation of N-Methyl-D-Aspartate receptor results in the production of ROS, which appears to be critical for synaptic plasticity, one of the cellular mechanisms that underlie learning and memory. In this work, we studied the effect of iron in the activation of MAPK/ERK pathway and on Ca2+ signaling in neuronal PC12 cells. We found that iron-dependent generation of hydroxyl radicals is likely to modulate Ca2+ signaling through RyR calcium channel activation, which, in turn, activates the MAPK/ERK pathway. These findings underline the relevance of iron in normal neuronal function. <![CDATA[Regulation of transepithelial transport of iron by hepcidin]]> Hepcidin (Hepc) is a 25 amino acid cationic peptide with broad antibacterial and antifungal actions. A likely role for Hepc in iron metabolism was suggested by the observation that mice having disruption of the gene encoding the transcription factor USF2 failed to produce Hepc mRNA and developed spontaneous visceral iron overload. Lately, Hepc has been considered the "stores regulator," a putative factor that signals the iron content of the body to intestinal cells. In this work, we characterized the effect of Hepc produced by hepatoma cells on iron absorption by intestinal cells. To that end, human Hepc cDNA was cloned and overexpressed in HepG2 cells and conditioned media from Hepc-overexpressing cells was used to study the effects of Hepc on intestinal Caco-2 cells grown in bicameral inserts. The results indicate that Hepc released by HepG2 inhibited apical iron uptake by Caco-2 cells, probably by inhibiting the expression of the apical transporter DMT1. These results support a model in which Hepc released by the liver negatively regulates the expression of transporter DMT1 in the enterocyte <![CDATA[<strong>Heme oxygenase 1 overexpression increases iron fluxes in Caco-2 cells</strong>]]> Heme oxygenase-1 is a microsomal enzyme that, when induced by stress, protects the cells from oxidative injury. Heme oxygenase-1 participates in the cleavage of the heme ring producing biliverdin, CO and ferrous Fe. The released Fe becomes part of intracellular Fe pool and can be stored in ferritin or released by an iron exporter. The mechanism by which heme enters cells is not completely understood, although it had been suggested that it might be internalized by an endocytosis process. In this study, we expressed a full-length Heme oxygenase-1 cDNA in Caco-2 cells and measured intracellular iron content, heme-iron uptake and transport and immunolocalization of heme oxygenase-1 in these cells. We found that heme oxygenase-1 expressing cells showed increased apical heme iron uptake and transepithelial transport when compared to control cells. These results suggested that heme oxygenase-1 mediates heme iron influx and efflux in intestinal cells. <![CDATA[Characterization of mitochondrial iron uptake in HepG2 cells]]> There is increasing evidence that accumulation of redox-active iron in mitochondria leads to oxidative damage and contributes to various neurodegenerative diseases, such as Friedreich's ataxia and Parkinson's disease. In this work, we examined the existence of regulatory mechanisms for mitochondrial iron uptake and storage. To that end, we used rhodamine B- [(1,10-phenanthrolin-5-yl)amino carbonyl ] benzyl ester, a new fluorescent iron-sensitive probe that is targeted specifically to the mitochondrion. We found that extracellular iron was incorporated readily into mitochondria in an apparently saturable process. Moreover, the rate of iron incorporation responded to the Fe status of the cell, an indication that the mitochondrion actively regulates its iron content.