Iron-deficient Rats Research Articles

Anti-anemic potential of Eruca sativa L. in iron-deficient rat model; network pharmacology profiling.

Iron deficiency anemia is a global health concern, affecting around 2 billion people. Oral iron therapy often causes severe gastro-intestinal issues. Eruca sativa, member of the Brassicaceae family, is valued in traditional medicine and renowned for its rich iron and vitamin C content. This study aims to evaluate the anti-anemic properties of E. sativa extract invivo and identify its compounds targeting anemia mechanisms using network pharmacology. Thirty-two Sprague-Dawley rats (200 ± 250 g) were split into two distinct groups, iron-deficient and iron-sufficient. Three different doses (200, 400, and 800 mg/kg) of aqueous extract of E. sativa were checked against anemia by studying hematological, oxidative stress, and histopathological parameters. GC-MS analysis of E. sativa revealed its phytochemical profile, followed by ADME screening. Network pharmacology explored targets related to iron deficiency anemia, with oral bioavailability and drug likeness assessment for compounds. The administration of extracts significantly improved various blood parameters, including osmotic fragility, Hb, RBCs, MCV, PCV, and alkaline phosphatase; catalase activity; and histopathological parameters such as liver in both iron-deficient and iron-sufficient rats (p < .001). Seventy-nine compounds were identified in E. sativa aqueous extract, with only six of them found to be bioavailable and drug-like against multiple targets. Gene ontology and pathway analysis revealed their diverse molecular, biological, and cellular functions. One gene EGFR was found to have functional association with ID anemia, suggesting potential for using E. sativa extracts. The study concludes that E. sativa extract has potential for iron deficiency anemia treatment, offering hope for future pharmaceutical interventions.

Identification of Genes Responding to Iron or Choline Treatment for Early-Life Iron Deficiency in the Male Rat Hippocampal Transcriptomes

BackgroundDevelopmental iron deficiency (ID) is associated with long-term cognitive and affective behavioral impairments in humans. Preclinical studies have shown that developmental ID has short- and long-term effects on gene regulation. Prenatal choline supplementation partially rescues early-life ID-induced cognitive deficits in adult male rats. ObjectivesTo identify acute and long-term changes in biological processes regulated by developmental ID and modifiable by choline. MethodsThis study compares the hippocampal transcriptomes of postnatal day (P) 15 iron-deficient (acute) and P65 formerly ID (persistent) rats with or without prenatal choline treatment. Pregnant rats were fed an ID (4 mg/kg Fe) or iron-sufficient (IS) (200 mg/kg Fe) diet from gestational day (G) 2 to P7 with or without choline supplementation (5 g/kg choline) from G11 to G18. Hippocampi were collected from P15 or P65 offspring and analyzed for gene expression by RNA sequencing. ResultsDevelopmental ID-induced changes suggested modified activity of oxidative phosphorylation and fatty acid metabolism. Prenatal choline supplementation induced robust changes in gene expression, particularly in iron-deficient animals, where it partially mitigated the early-life ID-dysregulated genes. Choline supplementation also altered the hippocampal transcriptome in the IS rats, with indications for both beneficial and adverse effects. ConclusionsThis study provided global assessments of gene expression regulated by iron and choline. Our new findings highlight genes responding to iron or choline treatments, including a potentially novel choline-regulated transporter (IPO7), with shared effects on neuroinflammation in the male rat hippocampus.

Differences in iron balance observed with dietary cholic acid supplementation and marginal iron deficiency in rats.

We investigated whether a cholic acid (CA)-supplemented diet and marginal iron deficiency (MID) diet influence hepatic lipid accumulation and iron balance in rats for 2 weeks. The CA diet enhanced hepatic lipid accumulation and modulated iron metabolism such as enhancement of fecal iron excretion, reduction in iron absorption, and no alteration in plasma iron levels. The MID diet did not alter hepatic lipid concentrations with reduced iron concentration in the liver and plasma. In combination, influence of the CA supplementation on the hepatic iron concentration was opposite between iron-sufficient and MID conditions. In the liver, the CA diet enhanced lipocalin 2 expression, whereas the MID diet enhanced transferrin receptor 1 expression and reduced hepcidin expression. This study revealed an involvement of 12-hydroxylated bile acids in regulation of hepatic iron concentration under MID condition.

Flammulina velutipes polysaccharide-iron(III) complex used to treat iron deficiency anemia after being absorbed via GLUT2 and SGLT1 transporters

Iron deficiency anemia (IDA) is a common nutritional problem, but traditional iron supplements cause many adverse reactions. Thus, the development of a novel iron supplement might be significant for the treatment of IDA. This study aimed to study the transport mechanism of Flammulina velutipes polysaccharide-iron complex (FVP1-Fe(III)) in Caco-2 cells and the therapeutic effect on IDA rats, as well as the influence on gut microbiota in vivo. These results showed that in vitro, the uptake of FVP1-Fe(III) was mediated by sodium-dependent glucose transporter-1 (SGLT1) and facilitated glucose transporter-2 (GLUT2) and GLUT2 played a dominant function. The multidrug resistance-associated protein-2 (MRP-2) was involved in the efflux of FVP1-Fe(III) across the Caco-2 cells. In vivo, FVP1-Fe(III) had a better restorative effect on blood parameters and iron status indicators in rats with IDA as compared with FeSO4 and exerted this effect by downregulating the expression of hepcidin. FVP1-Fe(III) could also regulate gut microbiota dysbiosis in iron deficiency rats by returning the relative abundance of gut microbiota to the normal level. Besides, as a dietary factor, vitamin C (vit C) could enhance the therapeutic effect of FVP1-Fe(III). These present findings showed that FVP1-Fe(III) could be exploited as a novel iron supplement to treat IDA.

Classical Selenium-Dependent Glutathione Peroxidase Expression Is Decreased Secondary to Iron Deficiency in Rats

While there are reports that classical selenium-dependent glutathione peroxidase (Se-GPX1) activity is decreased during iron deficiency, the relationship between tissue iron status and Se-GPX1 activity remains speculative. This study was undertaken to investigate the mechanism for the decrease in Se-GPX1 activity during iron deficiency. Male weanling Sprague-Dawley rats were given free access to either an iron-deficient or an iron-adequate diet for eight weeks, after which blood, livers, kidneys, hearts, brains and testes were surgically excised. During iron deficiency, Se-GPX1 mRNA levels in liver tissue were decreased by approximately 55%. Similarly, the concentration of immunoreactive Se-GPX1 protein and total selenium-dependent glutathione peroxidase (Se-GPX) activity were decreased by 55% and 60%, respectively. In kidney, heart and brain total Se-GPX activities were depressed as much as 33%. Selenium concentration in liver was reduced by 42%, whereas the decrease in Se concentrations in kidney, heart, and brain ranged from 17 to 25%. Concentrations of plasma Se also were reduced by 18%, but testes showed little change in either Se-GPX activity or Se concentration during iron deficiency. Results suggest that the synthesis of Se-GPX1 protein is decreased during iron deficiency possibly due to pretranslational regulation.

True Absorption and Retention of Supplemental Iron is More Efficient when Iron is Administered every Three Days rather than Daily to Iron-Normal and Iron-Deficient Rats

Absorption of daily iron supplements is inefficient. Detailed absorption patterns of FeSO4 supplements simulating World Health Organization-recommended doses were studied in iron-deficient and iron-normal male Sprague-Dawley rats. For 12 d after weaning, the rats were fed a premeal containing <20 (iron-deficient group) or 400 μg Fe (iron-normal group) twice daily followed by iron-free AIN-76 diet for 1 h. Then, both groups (iron-deficient and -normal) were divided in three groups, one continuing without change and two receiving 4000 μg of premeal Fe either daily or every 3 d to match intestinal mucosa renewal time. Food intake, growth, hemoglobin concentration and periodic 59Fe-labeled iron absorption and rate of loss were determined. Iron-deficient rats became anemic, and ate and grew less. Iron absorption was stable in normal (34.2%), deficient (89.7%) and normal, intermittently supplemented (9.5%) groups. Absorption decreased logarithmically in daily supplemented rats, whereas in iron-deficient, intermittently supplemented rats absorption decreased slowly and linearly. Rates of iron loss were significantly accelerated in daily supplemented rats. Thirteen-day total iron retention in intermittently supplemented normal and deficient rats was 62 and 86%, respectively, of that of daily supplemented rats. Iron supplementation timed to match mucosal renewal is more efficient.

Brain Iron, Transferrin and Ferritin Concentrations Are Altered in Developing Iron-Deficient Rats

To study the iron, transferrin, and ferritin distribution at subcellular levels in response to acute dietary iron deficiency, we tested the hypothesis that early post-weaning iron deficiency can change iron and iron regulatory protein concentrations in rat brain. Male Sprague-Dawley rats were fed diets containing either 2 or 35 µg iron/g for 2, 3 or 4 wk starting at 21 d of age. Brain iron, transferrin and ferritin concentrations in cytosolic and microsomal fractions of either whole brain or pons and cerebellum were then determined. After 14 d of dietary iron restriction, brain iron concentrations were 50% lower in the microsomal fraction and 30% lower in cytosol compared with controls. Brain cytosolic transferrin concentration almost doubled in the same animals. Brain ferritin concentration in fractions from rats fed the iron-deficient diet for 14 d was lower than in controls, but then remained fairly constant. Absolute brain weight and total brain protein contents were unaffected by iron restriction. This study extends previous research by demonstrating that the brain responds to changes in body iron status with a change in transferrin concentration. If the dietary restriction is quite severe, this adaptation is insufficient. This study also notes that brain ferritin decreases with decreasing body iron status, though it was less responsive than nonheme iron in liver. The concept that iron enters the brain through a highly regulated endocytotic process at the blood brain barrier, that undoubtedly involves the regulation of transferrin receptors in capillary endothelial cell, is supported by our observation of elevated transferrin concentrations in brain of iron-deficient rats.

H-ferritin in sows' colostrum- and milk-derived extracellular vesicles: a novel iron delivery concept.

Iron deficiency anemia is a significant problem in piglets, as they are born with insufficient iron stores for supporting their rapid body growth. Further, sows' milk contains inadequate iron levels for meeting the demands of piglet rapid growth in the pre-wean stage. The forms of iron present in the milk are essential to understanding bioavailability and potential routes for supplementing iron to mitigate iron deficiency anemia in piglets. Recently, our studies showed that H-ferritin (FTH1) is involved in iron transport to different tissues and can be used as an oral iron supplement to correct iron deficiency in rats and monkeys. In this study, we investigate the FTH1 levels in colostrum and milk in Yorkshires-crossbred sows (n = 27) and collected samples at the 1st, 15th, and 28th days of lactation to measure FTH1. Colostrum and milk were found to have FTH1, but there is no significant difference between the different days of lactation. FTH1 has been observed to be enriched in extracellular vesicles (EVs) of other species, and therefore examined the EVs in the samples. Colostrum-derived EVs were enriched with L-ferritin compared to FTH1, while in milk-derived EVs, only FTH1 was detected (P = 0.04). In milk-derived EVs, FTH1 was significantly higher (P = 0.021; P = 006) than FTH1 in colostrum-derived EVs. Furthermore, FTH1 levels of milk-derived EVs were significantly higher (P = 0.0002; P = 0004) than whole milk and colostrum FTH1. These results indicate that FTH1 is enriched in the milk-derived EVs and suggest that EVs play a predominant role in the FTH1 delivery mechanism for the piglet. The extent to which FTH1 in EVs accounts for the overall iron delivery mechanism in piglets is yet to be determined.

Effects of Iron Supplementation on Testicular Function and Spermatogenesis of Iron-Deficient Rats.

Iron deficiency is the most common micronutrient deficiency in the world. Previous studies have shown that iron deficiency increases oxidative stress and decreases antioxidant enzymes, and studies of male infertility indicated that oxidative stress may affect male reproductive functions. The aim of this study was to investigate the effects of iron supplementation on spermatogenesis and testicular functions in iron-deficient rats. Three-week-old male Sprague Dawley (SD) rats were randomly divided into two groups: an iron-adequate control (AI group, 35 ppm FeSO4) and an iron-deficient group (ID group, <5 ppm FeSO4). After three weeks, the iron-deficient group was divided into an original iron-deficient group and five iron-supplemented groups, the latter fed diets containing different doses of FeSO4 (6, 12, 18, 24, and 35 ppm). After five weeks, blood and testis tissue were analyzed. We presented as median (interquartile range, IQR) for continuous measurements and compared their differences using the Kruskal–Wallis test followed by the Mann–Whitney U test among groups. The results showed that as compared with the AI group, the ID group had significantly lower serum testosterone and poorer spermatogenesis (The medians (QR) were 187.4 (185.6–190.8) of AI group vs. 87.5 (85.7–90.4) of ID group in serum testosterone, p < 0.05; 9.3 (8.8–10.6) of AI group vs. 4.9 (3.4–5.4) of ID group in mean testicular biopsy score (MTBS], p < 0.05); iron supplementation reversed the impairment of testis tissue. In the testosterone biosynthesis pathway, iron supplementation improved the lowered protein expressions of hydroxysteroid dehydrogenases caused by iron deficiency. Additionally, decreased activities of glutathione peroxidase and catalase, and increased cleaved-caspase 8 and caspase 3 expression, were found in the iron-deficient rats. The iron-supplemented rats that received > 12 ppm FeSO4 exhibited improvements in antioxidant levels. In conclusion, iron supplementation can abrogate testis dysfunction due to iron deficiency through regulation of the testicular antioxidant capacity.

Striatal mechanism of the restless legs syndrome.

Brain iron deficiency has been reported to be associated with the restless legs syndrome (RLS). However, 30%-50% of RLS patients do not respond to iron therapy, indicating that mechanisms other than brain iron deficiency may also participate in this disease. The striatum is known to be involved in the modulation of motor activity. We speculated that dysfunction of the striatum may induce RLS. Two groups, wild-type (WT) and iron-deficient (ID) rats were used. Each group was divided into two subgroups, control and N-methyl-d-aspartate striatal-lesioned. After baseline recording, striatal-lesioned wild-type (WT-STL) and striatal-lesioned iron-deficient (ID-STL) rats were given pramipexole and thioperamide injections. Iron-deficient and ID-STL rats were then given a standard rodent diet for 4 weeks, and their sleep and motor activity were recorded. WT-STL rats showed periodic leg movements (PLM) in wake, an increase in PLM in slow wave sleep (SWS), a decrease in rapid-eye-movement sleep, and a decrease in the daily average duration of episodes in SWS. The sleep-wake pattern and motor activity did not differ between ID and ID-STL rats. Thioperamide or pramipexole injection decreased PLM in sleep and in wake in WT-STL rats and ID-STL rats. Unlike ID rats, whose motor hyperactivity can be reversed by iron replacement, PLM in wake and in sleep in ID-STL rats were not fully corrected by iron treatment. Lesions of the striatum generate RLS-like activity in rats. Dysfunction of the striatum may be responsible for failure to respond to iron treatment in some human RLS patients.

Gestational and Lactational Iron Deficiency Anemia Impairs Myelination and the Neurovascular Unit in Infant Rats.

Iron deficiency anemia is a prevalent health problem among pregnant women and infants, particularly in the developing countries that causes brain development deficits and poor cognitive outcomes. Since tissue iron depletion may impair myelination and trigger cellular hypoxic signaling affecting blood vessels, we studied myelination and the neurovascular unit (NVU) in infant rats born to mothers fed with an iron deficient (ID) or control diet from embryonic day 5 till weaning. Blood samples and brains of rat pups at postnatal day (PND) 14 and 30 were analyzed. PND 14 ID rats had severe microcytic hypochromic anemia that was almost reversed at PND 30 although hypomyelination and astrocyte immature phenotype in the corpus callosum were significant at that age. In CA1 hippocampal region, PND 14 and PND 30 ID rats showed significant reduced expression of the receptor β of the platelet-derived growth factor localized in pericytes and associated to aquaporin 4 (AQP4) immunopositive capillaries. Shorter AQP4 + capillaries and reduced AQP4 expression were also evidenced in PND 14 and PND 30 ID rats. In addition, pericyte membrane permeability through large-pore channels was transiently increased in ID rats at PND 14 but not at PND 30, while the blood-brain barrier permeability was not affected. Remarkably, transient increased pericyte permeability found in PND 14 ID rats was not directlyrelated to iron depletion, suggesting the involvement of other iron deficiency anemia-induced mechanisms. In summary, severe ID during gestation and lactation produces persistent hypomyelination and significantly affects hippocampal pericytes and astrocytes in the NVU which may trigger impaired neurovascular function.

Choline Supplementation Partially Restores Dendrite Structural Complexity in Developing Iron-Deficient Mouse Hippocampal Neurons

BackgroundFetal-neonatal iron deficiency causes learning/memory deficits that persist after iron repletion. Simplified hippocampal neuron dendrite structure is a key mechanism underlying these long-term impairments. Early life choline supplementation, with postnatal iron repletion, improves learning/memory performance in formerly iron-deficient (ID) rats. ObjectivesTo understand how choline improves iron deficiency–induced hippocampal dysfunction, we hypothesized that direct choline supplementation of ID hippocampal neurons may restore cellular energy production and dendrite structure. MethodsEmbryonic mouse hippocampal neuron cultures were made ID with 9 μM deferoxamine beginning at 3 d in vitro (DIV). At 11 DIV, iron repletion (i.e., deferoxamine removal) was performed on a subset of ID cultures. These neuron cultures and iron-sufficient (IS) control cultures were treated with 30 μM choline (or vehicle) between 11 and 18 DIV. At 18 DIV, the independent and combined effects of iron and choline treatments (2-factor ANOVA) on neuronal dendrite numbers, lengths, and overall complexity and mitochondrial respiration and glycolysis were analyzed. ResultsCholine treatment of ID neurons (ID + Cho) significantly increased overall dendrite complexity (150, 160, 180, and 210 μm from the soma) compared with untreated ID neurons to a level of complexitythat was no longer significantly different from IS neurons. The average and total length of primary dendrites in ID + Cho neurons were significantly increased by ∼15% compared with ID neurons, indicating choline stimulation of dendrite growth. Measures of mitochondrial respiration, glycolysis, and ATP production rates were not significantly altered in ID + Cho neurons compared with ID neurons, remaining significantly reduced compared with IS neurons. Iron repletion significantly improved mitochondrial respiration, ATP production rates, overall dendrite complexity (100–180 μm from the soma), and dendrite and branch lengths compared with untreated ID neurons. ConclusionsBecause choline partially restores dendrite structure in ID neurons without iron repletion, it may have therapeutic potential when iron treatment is not possible or advisable. Choline's mechanism in ID neurons requires further investigation.

Internalization and Decrease of Duodenal DMT1 Involved in Transient Suppression of Iron Uptake in Short-Acting Mucosal Block.

Mucosal block (MB) is induced by the oral administration of excess iron (10mg) and suppresses intestinal iron absorption for 3-72h. The inhibition of iron absorption is accompanied by the downregulation of molecules associated with intestinal iron absorption. Recently, we found that a smaller amount of iron (1mg) also induced a transient suppression of iron uptake without affecting gene expression levels (short-acting mucosal block, SAMB), which is specific to iron-deficient rats. In this study, we investigated how the nonheme iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) are involved in the transient suppression of iron uptake in SAMB. To induce SAMB, a test solution containing 1mg iron was infused into the duodenum loop in iron-sufficient and iron-deficient rats. Total duodenal DMT1 and DMT1-IRE expression were increased during iron deficiency. After 15min of 1mg iron loading, the fluorescence intensity of duodenal DMT1 in iron-deficient rats was decreased and was comparable to that in iron-sufficient rats. Internalized DMT1-IRE as puncta was observed at 15 and 60min after 1mg iron loading, and the number of punctas was significantly increased after 60min compared with control. There was no effect of 1mg iron loading on the intracellular distribution of duodenal FPN. Our results suggest that the decrease and internalization of DMT1-IRE protein may be related, at least in part, to iron uptake suppression in SAMB.

Genotype Score for Iron Status Is Associated with Muscle Fiber Composition in Women.

Human muscle fiber composition is heterogeneous and mainly determined by genetic factors. A previous study reported that experimentally induced iron deficiency in rats increases the proportion of fast-twitch muscle fibers. Iron status has been reported to be affected by genetic factors. As the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms are strongly associated with iron status in humans, we hypothesized that the genotype score (GS) based on these polymorphisms could be associated with the muscle fiber composition in humans. Herein, we examined 214 Japanese individuals, comprising of 107 men and 107 women, for possible associations of the GS for iron status with the proportion of myosin heavy chain (MHC) isoforms (I, IIa, and IIx) as markers of muscle fiber composition. No statistically significant correlations were found between the GS for iron status and the proportion of MHC isoforms in all participants. When the participants were stratified based on sex, women showed positive and negative correlations of the GS with MHC-IIa (age-adjusted p = 0.020) and MHC-IIx (age-adjusted p = 0.011), respectively. In contrast, no correlation was found in men. In women, a 1-point increase in the GS was associated with 2.42% higher MHC-IIa level and 2.72% lower MHC-IIx level. Our results suggest that the GS based on the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms for iron status is associated with muscle fiber composition in women.

Evaluation of candidate reference genes for quantitative real-time PCR analysis in a male rat model of dietary iron deficiency

BackgroundQuantitative real-time polymerase chain reaction (qPCR) is a reliable and efficient method for quantitation of gene expression. Due to the increased use of qPCR in examining nutrient-gene interactions, it is important to examine, develop, and utilize standardized approaches for data analyses and interpretation. A common method used to normalize expression data involves the use of reference genes (RG) to determine relative mRNA abundance. When calculating the relative abundance, the selection of RG can influence experimental results and has the potential to skew data interpretation. Although common RG may be used for normalization, often little consideration is given to the suitability of RG selection for an experimental condition or between various tissue or cell types. In the current study, we examined the stability of gene expression using BestKeeper, comparative delta quantitation cycle, NormFinder, and RefFinder in a variety of tissues obtained from iron-deficient and pair-fed iron-replete rats to determine the optimal selection among ten candidate RG.ResultsOur results suggest that several commonly used RG (e.g., Actb and Gapdh) exhibit less stability compared to other candidate RG (e.g., Rpl19 and Rps29) in both iron-deficient and iron-replete pair-fed conditions. For all evaluated RG, Tfrc expression significantly increased in iron-deficient animal livers compared to the iron-replete pair-fed controls; however, the relative induction varied nearly 4-fold between the most suitable (Rpl19) and least suitable (Gapdh) RG.ConclusionThese results indicate the selection and use of RG should be empirically determined and RG selection may vary across experimental conditions and biological tissues.

Basal and resistance exercise-induced increase in protein synthesis is impaired in skeletal muscle of iron-deficient rats

ObjectivesWe aimed to investigate the effect of iron deficiency on basal- and contraction-induced increases in muscle protein synthesis. MethodsFour-wk-old male Sprague–Dawley rats were divided into three groups. The rats in two of the three groups had free access to a control diet (AD) or iron-deficient diet (ID) for 4 wk. The rats in the third group (CON) were pair-fed the control diet to the mean intake of the ID group. ResultsIn comparison with the CON group, the ID group showed significantly lower hematocrit and hemoglobin concentrations, iron-containing protein levels, and total iron content in skeletal muscle, but non-iron-containing protein levels did not show any differences between the groups. Protein synthesis, measured by puromycin-labeled peptides, was lower in the ID group compared with the CON group in both basal- and contraction-stimulated states. The ID diet impaired the activation levels of signaling pathways involved in protein synthesis, such as ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1. Furthermore, dietary iron deficiency decreased autophagy capacity, but did not affect the ubiquitinated protein content. ConclusionsThese results suggest that severe iron deficiency decreases not only basal but also muscle contraction-induced increases in protein synthesis due to, at least in part, downregulation of the protein synthesis signaling pathway in the skeletal muscle.

Insights image for "Evaluation of angiogenic signaling molecules associated with reactive thrombocytosis in an iron deficient rat model".

Insights image for "Evaluation of angiogenic signaling molecules associated with reactive thrombocytosis in an iron deficient rat model".

Dietary Iron Intake in Excess of Requirements Impairs Intestinal Copper Absorption in Sprague‐Dawley Rat Dams, Causing Copper Deficiency in Neonatal Pups

Consumption of a high-iron diet by previously iron-deficient rat dams causes copper insufficiency; however, pathophysiological outcomes in their offspring are unknown. We hypothesized that high-iron consumption by dams would also lead to copper depletion in suckling pups. Accordingly, 8-9-week-old, pregnant Sprague-Dawley rats were randomized and initially fed a low-Fe diet for ≍7 days (to induce iron deficiency), and then switched to one of five AIN-93G-based diets, with variable iron levels (normal [80 ppm] to high [up to 1600 ppm]) and marginal copper content (2 ppm; normal is 6-7 ppm). Dams were fed these diets throughout the remainder of pregnancy and during lactation, until the termination of the experiments (when suckling pups were 13-15 days of age). Subsequently, hematological parameters and serum and tissue iron concentrations were measured, and copper biomarker analyses were performed. Intestinal 64Cu absorption and tissue distribution in dams, and 64Cu delivery to the suckling pups, were also assessed. Results showed that serum para-phenylenediamine (pPD) oxidase activity (reflecting serum ceruloplasmin [Cp]) was suppressed (p<0.05) in dams consuming 5-fold excess iron and in pups suckling dams that consumed iron at 10-fold excess. Hepatic nonheme iron concentrations increased in dams and pups (p<0.05) as dietary iron levels increased (possibly due to inhibition of Cp activity, which is required for iron release from hepatocytes). When dams consumed iron at 20-fold above requirements, pup growth was impaired (p<0.05), possibly due to copper depletion, as also indicated by decreases in erythrocyte SOD activity (which accurately reflects copper status) (p<0.05). Moreover, in iron-supplemented dams, intestinal copper (64Cu) absorption and 64Cu activity in blood, liver, and spleen were all decreased (p<0.05). A similar trend was also noted in pups suckling dams consuming exces iron, with 64Cu accumulation in blood, liver, and spleen (p<0.05) being lower than in controls. We conclude that maternal intake of excessive iron (~10-fold above the dietary recommendation) impairs absorption of dietary copper and concomitantly depletes copper in neonatal pups. Low milk copper content was the most likely explanation for this outcome, although this remains to be experimentally proven.

Diet-induced iron deficiency in rats impacts small intestinal calcium and phosphate absorption.

Recent reports suggest that iron deficiency impacts both intestinal calcium and phosphate absorption, although the exact transport pathways and intestinal segment responsible have not been determined. Therefore, we aimed to systematically investigate the impact of iron deficiency on the cellular mechanisms of transcellular and paracellular calcium and phosphate transport in different regions of the rat small intestine. Adult, male Sprague-Dawley rats were maintained on a control or iron-deficient diet for 2weeks and changes in intestinal calcium and phosphate uptake were determined using the in situ intestinal loop technique. The circulating levels of the hormonal regulators of calcium and phosphate were determined by ELISA, while the expression of transcellular calcium and phosphate transporters, and intestinal claudins were determined using qPCR and western blotting. Diet-induced iron deficiency significantly increased calcium absorption in the duodenum but had no impact in the jejunum and ileum. In contrast, phosphate absorption was significantly inhibited in the duodenum and to a lesser extent the jejunum, but remained unchanged in the ileum. The changes in duodenal calcium and phosphate absorption in the iron-deficient animals were associated with increased claudin 2 and 3 mRNA and protein levels, while levels of parathyroid hormone, fibroblast growth factor-23 and 1,25-dihydroxy vitamin D3 were unchanged. We propose that iron deficiency alters calcium and phosphate transport in the duodenum. This occurs via changes to the paracellular pathway, whereby upregulation of claudin 2 increases calcium absorption and upregulation of claudin 3 inhibits phosphate absorption.

Evaluation of angiogenic signaling molecules associated with reactive thrombocytosis in an iron-deficient rat model.

Iron deficiency anemia (IDA)-induced reactive thrombocytosis can occur in children and adults. The underlying mechanism for this phenomenon is indeterminate. Traditional cytokines such as thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 involved in megakaryopoiesis have not been shown to be the cause. Recent studies suggest that growth factors and signaling molecules involved with angiogenesis influence the proliferation and differentiation of megakaryocytes. We investigated the possible association between angiogenic cytokines with reactive thrombocytosis due to IDA in an iron-deficient(ID) rat model. Complete blood count, iron panels, and TPO levels were measured at baseline and 5 weeks later in both control (C) and ID rats. Angiogenic cytokines were evaluated in the bone marrow in all rats. We successfully induced IDA in our rats by phlebotomy and reduced iron diet. We did not find an increase of TPO in ID rats. A review of the bone marrow showed an increase in the number of megakaryocytes, vascular structures, as well as increased intensity of stain forvascular endothelial growthfactor (VEGF), and CXCchemokine receptor4(CXCR4) in rats with IDA compared to controls. Our results of histological bone marrow data suggest an important role for angiogenesis in the development of IDA-induced thrombocytosis. Thrombocytosis is common with IDA in both children and adults, but the mechanism is unclear. We confirmed that TPO is not the major driver of iron deficiency-associated thrombocytosis. We confirmed the increase in the number of megakaryocytes in the bone marrow despite stable TPO levels. We provided evidence supporting an important role of angiogenesis in megakaryocytopoiesis/thrombopoiesis with increased vascular structures and angiogenic cytokines in the bone marrow of iron-deficient rats. The demonstration that angiogenesis may play an important role in secondary thrombocytosis could lead to a new approach in treating symptomatic reactive thrombocytosis by targeting angiogenesis.