The influence of dietary iron on zinc in rat

The purpose of this study was to clarify the influence of iron on zinc status. The animals were divided into four groups, consisting of five rats in each group. The control group was fed on basal diet with adequate levels of zinc and iron, whereas the experimental group was fed diets containing different levels of iron ad libitum for 15 d. Low levels of iron (LFe) significantly increased the zinc absorption percentage but there was a decrease in high (HFe) and very high iron (VHFe) level groups (p<0.001). The retention percentage changes were found to be parallel to the changes in the absorption percentage curve. It was found that zinc (per total dry tissue) and Zn-65 (per total tissue) increased in the rats fed the LFe, whereas in general they decreased in the rats fed the HFe and VHFe diets. Significant changes were found in the duodenum and liver. Zn-65 (per g wet tissue) significantly increased in the brain and liver in the LFe group, but there was a decrease in the duodenum, ileum, kidney, liver, and brain in the HFe and VHFe groups. Changes in the level of zinc (per g dried tissue) were found to be parallel to the changes in Zn-65 in all the groups. The dietary proportions of iron appear to influence zinc metabolism at the intestinal and cellular transport levels over a given period of time.     

The effect of various dietary zinc concentrations on the biological interactions of zinc,copper, and iron in rats

Three groups (14 rats each) were fed one of the following diets for 8 wks: a control purified basal diet containing 12 ppm zinc, 5 ppm copper, and 35 ppm iron; the basal diet with less than 2 ppm zinc; or the basal diet supplemented with 1000 ppm zinc. Rats fed the zinc-deficient diet had decreased weight gain, moderate polydipsia, and intermittent mild diarrhea. The zinc-supplemented rats had a cyclical pattern of food intake and weight loss from weeks 5 to 8. Tissue concentrations suggest that zinc and copper were not mutually antagonistic with chronic dietary imbalances. If tissue element concentrations reflected intestinal uptake, then competition and/or inhibition of intestinal uptake occurred between zinc and iron. The fluctuations in tissue element concentrations that occurred with increased duration of the study were at variance with previous studies of shorter time periods. The dietary proportions of zinc, copper, and iron appear to influence zinc, copper, and iron metabolism at the intestinal and cellular transport levels over a given period of time.     

Effect of dietary iron deficiency on mineral levels in tissues of rats

To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats.     

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.     

Modulation of aconitase, metallothionein, and oxidative stress in zinc-deficient rat intestine during zinc and iron repletion

Potential interactions between zinc and iron during absorption and its functional consequences on intestinal oxidative damage and antioxidant status were studied using the zinc-deficient rat as a model. Zinc depletion produced mild-moderate iron deficiency in addition to zinc deficiency, which could be corrected by repletion with iron and zinc. The localization and intensity of both iron and zinc in the intestinal mucosa showed a pronounced decrease in the presence of the other metal, indicating negative interactions. Zinc-deficient intestine exposed to iron alone exhibited elevated peroxidative damage and compromised functional integrity, despite increased expression of ferritin. Inclusion of zinc significantly reduced the damage and improved the functional integrity, accompanied by decreased expression of ferritin. Decreased expression of ferritin in the presence of zinc was consistent with reduced aconitase activity, suggesting its modulation by zinc. Further, inclusion of iron along with zinc was associated with induction of ferritin and metallothionein in tune with the amount of iron and zinc localized in the intestinal mucosa, respectively. These results suggest that zinc and iron interact negatively with cytosolic aconitase, but prove beneficial in reducing the oxidative stress, apart from improving functional integrity and iron/zinc status.     

The effect of 1 alpha, 25-dihydroxycholecalciferol on iron metabolism

Chronic administration of hypercalcemic doses of 1 alpha, 25-dihydroxycholecalciferol to intact, vitamin-D repleted rats for 4 weeks, enhanced net intestinal absorption of iron and liver iron stores. Daily net iron and calcium absorptions were found to be significantly correlated in both control and treated rats. In duodenal loop experiments, pretreatment with 1 alpha, 25-dihydroxycholecalciferol reversed the adverse effect of high Ca/Fe ratio on iron absorption. The increased intestinal absorption of iron did not result in a change of serum iron levels nor of total iron binding capacity due to the enhanced incorporation of absorbed iron into liver ferritin. The curve of uptake of 59Fe into circulating red cells of treated rats suggested retarded release of the isotope from stores. The hypothesis is advanced that the systemic metabolic defect (tissue hypoxia, raised erythropoietin levels) produced by 1 alpha, 25-dihydroxycholecalciferol is responsible for the disruption of the physiological coordination between iron stores and intestinal absorption.     

Relationship between intestinal iron-transporter expression,hepatic hepcidin levels and the control of iron absorption

Hepcidin is an anti-microbial peptide predicted to be involved in the regulation of intestinal iron absorption. We have examined the relationship between the expression of hepcidin in the liver and the expression of the iron-transport molecules divalent-metal transporter 1, duodenal cytochrome b, hephaestin and Ireg1 in the duodenum of rats switched from an iron-replete to an iron-deficient diet or treated to induce an acute phase response. In each case, elevated hepcidin expression correlated with reduced iron absorption and depressed levels of iron-transport molecules. These data are consistent with hepcidin playing a role as a negative regulator of intestinal iron absorption.     

Intestinal iron absorption and mucosal transferrin in rats subjected to hypoxia

Three days hypoxia (0.5 atm) increased the haemoglobin and haematocrit values in rats paralleled by enhanced intestinal iron absorption. The destination of recently-absorbed iron was primarily the erythropoietic system, viz. bone marrow, spleen and red cells. Total plasma transferrin, was increased by 30%, but no significant changes in mucosal transferrin were found. No increase in labelling of mucosal transferrin by absorbed iron was observed. These results suggest that mucosal transferrin does not play a major role in the regulation of intestinal iron absorption in hypoxia.     

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.     

Dietary cadmium effect on iron metabolism in chickens

A control group of 1-day-old chicks, fed on commercial food, were compared with different experimental lots that had all received a supplement of 100 ppm Cd. The hematocrit, hemoglobin and ceruloplasmin concentrations, and metal contents (Fe, Cu, Zn, Cd) in plasma and in the liver were determined after either 4 or 9 weeks of treatment. The intestinal iron absorption and their ferrokinetics were also studied in 10-week-old Cd-fed chicks. The anemia-producing effect of cadmium was already evident after the second week of treatment. The iron supplement (oral or injected) corrected the anemia, but did not correct the depression of growth effect. Plasma iron was not affected, but the liver stores were reduced by 50%. Neither the plasma copper and ceruloplasmin, nor the copper content in liver, were affected. Zinc in the liver increased significantly (P<0.05). No statistical differences in plasma iron turnover were observed between the control and Cd-fed chicks, but the red blood cell utilization was higher (P<0.01) in Cd-fed groups. The intestinal iron absorption was clearly reduced (P<0.001) where cadmium was presented in the perfusion fluid “in vivo” experiments. This suggested that cadmium reduced the iron liver stores through its effect on intestinal iron absorption. However, it also seems that it did not interfere in iron mobilization, since the plasma iron was unaffected and the Cd-fed chicks presented increased plasma iron after estrogen administration. The indirect effect of cadmium on copper metabolism is uncertain.     

Tissues and organs as indicators of intestinal absorption of minerals and trace elements,evaluated in rats

Tissue and organ deposition and blood parameters were evaluated as indices of mineral and trace element absorption in rats. The absorption of elements was quantified in relation to nitrogen retention, i.e., considering the weight gain and new tissue synthesis. A rapeseed meal diet was supplied with three levels of calcium, two levels of zinc, and two levels of copper in a factorial design. In general, an increase in dietary mineral content increased the relative absorption, which in turn, increased the tissue deposition progressively. Striated muscle, however, did not respond to either an increased calcium or zinc supply. Furthermore, an increased calcium absorption caused a depression of the fractional phosphorus and magnesium content of femur bones. The copper content of the kidneys and the heart muscle was directly proportional to the amount of absorbed zinc and iron, respectively. The iron content of tissues was, in general, inversely proportional to zinc absorption and showed a tendency to be directly proportional to copper absorption. The zinc level in tissues was, in a similar way, inversely correlated to measured calcium absorption. In conclusion, interactions between elements do not only affect the intestinal element absorption, but also the distribution of already absorbed elements in tissues and organs.     

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0463-5) contains supplementary material, which is available to authorized users.     

Iron Excess Disturbs Metabolic Status and Relative Gonad Mass in Rats on High Fat, Fructose, and Salt Diets

The aim of this study was to assess the metabolic and physiological changes in rats fed a diet high in fat, fructose, and salt, and with excess iron level. Mineral status was also estimated. Wistar rats were assigned to groups fed either a standard control diet (C) or a diet high in fat, fructose, and salt. The noncontrol diets contained either normal (M) or high level (MFe) of iron. After 6 weeks, the length and weight of the rats were measured, and the animals were euthanized. The kidneys and gonads were collected, and blood samples were taken. Serum levels of insulin, nitric oxide, and iron were measured. The iron, zinc, copper, and calcium concentrations of tissues were determined. It was found that the M diet led to a significant increase in the relative kidney mass of the rats compared with the control group. Among the rats fed the M diet, markedly higher serum level of iron and lower levels of zinc and copper were observed in tissues, while significantly higher calcium levels were found in the gonads. The MFe diet resulted in decreased obesity index, insulin level, and nitric oxide serum concentration in the rats, when compared with both the M and C diets. The high iron level in the modified diet increased the relative mass of the gonads. The excess iron level in the diet disturbed the zinc, copper, and calcium status of tissues. The decrease in insulin and nitric oxide in rats fed the diet high in iron, fat, fructose, and salt was associated with disorders of zinc, copper, and calcium status, as well as with an increase in the relative mass of the gonads.     

Supplemental sodium phytate and microbial phytase influence iron availability in growing rats.

Five groups of individually housed albino rats (n = 7 each, initial average weight = 42 g) were fed diets based on corn starch and casein over a 4-week period. All diets were supplemented with 35 mg/kg of iron from FeSO4 x 7 H2O. Group I (control) was fed the basal diet free of phytic acid (PA) and phytase. By replacing corn starch by 7.5 g (groups II and IV) and 15 g phytic acid (groups III and V) from sodium phytate per kg diet, molar PA/iron ratios of 18 and 36 were obtained. In groups IV and V, 1000 U phytase from Aspergillus niger per kg diet were added. Food conversion efficiency ratio and growth rate as well as iron in plasma and spleen, hemoglobin, red blood cell count and erythrocyte zinc protoporphyrin were not influenced by the different dietary treatments. Dietary phytate reduced apparent iron absorption in groups II and III. Furthermore hematocrit, transferrin saturation and iron concentration in liver and femur were lowered in rats fed diets with PA, while total and latent iron-binding capacity of plasma increased. Microbial phytase supplementation (groups IV and V) partly counteracted the antinutritive effects of phytic acid on iron availability.     

Influence of dietary iron deficiency on acute metal intoxication

The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal and intestinal metallothionein (MT), disposition of the metals, and alterations in hematological parameters was investigated in rats. The administration of cadmium induced the hepatic, renal and intestinal MT while that of nickel or lead induced hepatic MT only. However, dietary iron deficiency did not influence the cadmium induced tissue MT but enhanced the ability of nickel or lead to restore the normal synthesis of renal and intestinal MT lowered under the influence of reduced body iron status. The accumulation of lead in liver and kidney and that of cadmium enhanced in liver only, while tissue deposition of nickel remained unaffected by iron deficiency. The induction of hepatic MT by three metals appears related to the concomitant rise in the hepatic zinc, calcium and iron levels in normal rats. However, dietary iron deficiency increased the hepatic zinc in response to nickel or cadmium and that of heptic calcium in response to lead.     

Vitamin B6 deficiency alters tissue iron concentrations in the Wistar rat

PurposeWe investigated the effect of a vitamin B6 deficiency and pair-feeding on tissue trace element status.MethodTissue zinc, copper and iron concentrations were measured in 3 groups of young, male Wistar rats receiving a diet of 3.5 mg/kg (control group), 0 mg/kg (deficient group) and a pair-fed group over 8 weeks. The pair-fed group received the same diet consumed by the control. Tissue trace element analysis was performed using atomic absorption spectrophotometry and plasma vitamin B6 status was determined using HPLC.ResultsDeficiency resulted in elevation in liver iron concentration and reduction in muscle iron concentration. Muscle copper concentrations were reduced in the pair-fed and deficient groups vs. the control group. Tissue zinc concentrations remained unaffected by the deficiency. Kidney iron and heart copper levels were elevated in the pair-fed group.ConclusionsThe liver and muscle iron changes were due to the deficiency and not to reduced calorie intake and the latter may be due to impaired heme synthesis. The differences in copper between the groups were due to reduced food intake. Zinc seems to form a fixed pool in these animals. A dietary deficiency of vitamin B6 impacts on the trace element status of certain tissues in key metabolic tissues and hence needs to be factored into the amelioration of the condition.     

Effect of dietary copper and zinc levels on tissue copper,zinc, and iron in male rats

The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 μg/g) and five levels of zinc (1, 4.5, 10, 100, 1000 μg/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 μg Cu, 100 μg Zn/g diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily when dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.