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This is the fifth in the series of reviews developed as part of the Biomarkers of Nutrition for Development (BOND) program. The BOND Iron Expert Panel (I-EP) reviewed the extant knowledge regarding iron biology, public health implications, and the relative usefulness of currently available biomarkers of iron status from deficiency to overload. Approaches to assessing intake, including bioavailability, are also covered. The report also covers technical and laboratory considerations for the use of available biomarkers of iron status, and concludes with a description of research priorities along with a brief discussion of new biomarkers with potential for use across the spectrum of activities related to the study of iron in human health.The I-EP concluded that current iron biomarkers are reliable for accurately assessing many aspects of iron nutrition. However, a clear distinction is made between the relative strengths of biomarkers to assess hematological consequences of iron deficiency versus other putative functional outcomes, particularly the relationship between maternal and fetal iron status during pregnancy, birth outcomes, and infant cognitive, motor and emotional development. The I-EP also highlighted the importance of considering the confounding effects of inflammation and infection on the interpretation of iron biomarker results, as well as the impact of life stage. Finally, alternative approaches to the evaluation of the risk for nutritional iron overload at the population level are presented, because the currently designated upper limits for the biomarker generally employed (serum ferritin) may not differentiate between true iron overload and the effects of subclinical inflammation. [ABSTRACT FROM AUTHOR]

Background: The role of the placenta in regulating micronutrient transport in response to maternal status is poorly understood.Objective: We investigated the effect of prenatal nutritional supplementation on the regulation of placental iron and zinc transport.Methods: In a randomized trial in rural Gambia [ENID (Early Nutrition and Immune Development)], pregnant women were allocated to 1 of 4 nutritional intervention arms: 1) iron and folic acid (FeFol) tablets (FeFol group); 2) multiple micronutrient (MMN) tablets (MMN group); 3) protein energy (PE) as a lipid-based nutrient supplement (LNS; PE group); and 4) PE and MMN (PE+MMN group) as LNS. All arms included iron (60 mg/d) and folic acid (400 μg/d). The MMN and PE+MMN arms included 30 mg supplemental Zn/d. In a subgroup of ∼300 mother-infant pairs, we measured maternal iron status, mRNA levels of genes encoding for placental iron and zinc transport proteins, and cord blood iron levels.Results: Maternal plasma iron concentration in late pregnancy was 45% and 78% lower in the PE and PE+MMN groups compared to the FeFol and MMN groups, respectively (P < 0.001). The mRNA levels of the placental iron uptake protein transferrin receptor 1 were 30-49% higher in the PE and PE+MMN arms than in the FeFol arm (P < 0.031), and also higher in the PE+MMN arm (29%; P = 0.042) than in the MMN arm. Ferritin in infant cord blood was 18-22% lower in the LNS groups (P < 0.024). Zinc supplementation in the MMN arm was associated with higher maternal plasma zinc concentrations (10% increase; P < 0.001) than in other intervention arms. mRNA levels for intracellular zinc-uptake proteins, in this case zrt, irt-like protein (ZIP) 4 and ZIP8, were 96-205% lower in the PE+MMN arm than in the intervention arms without added zinc (P < 0.025). Furthermore, mRNA expression of ZIP1 was 85% lower in the PE+MMN group than in the PE group (P = 0.003).Conclusion: In conditions of low maternal iron and in the absence of supplemental zinc, the placenta upregulates the gene expression of iron and zinc uptake proteins, presumably in order to meet fetal demands in the face of low maternal supply. The ENID trial was registered at www.controlled-trials.com as ISRCTN49285450. [ABSTRACT FROM AUTHOR]

Iron (Fe) metabolism is modified by many nutritional factors. Amino acids (AA) play a central role in various biological processes, such as protein synthesis and energy supply. However, the influence of AA status on iron metabolism has not been investigated. Here, we test whether AA alters iron metabolism in an intestinal cell model. Both Fe uptake and transfer across the cell monolayer were significantly increased by non-essential AA deficiency (both p < 0.001) while only Fe transfer was increased by essential AA deficiency (p < 0.0001). Both essential and non-essential AA deficiency decreased DMT1 (±IRE) exon1A mRNA expression (respectively p = 0.0007 and p = 0.006) and increased expression of ferritin heavy chain. DMT1 + IRE (also expressing exon1A or 1B) mRNA levels were decreased by essential AA deficiency (p = 0.012). The mRNA levels of total DMT1 were also decreased by essential, but not non-essential, AA deficiency (p = 0.006). Hepcidin levels were increased significantly by non-essential amino acid deprivation (p = 0.047). Protein levels of ferroportin and/or ferritin heavy chain were not altered by AA deficiency, suggesting that they had no effect on Fe efflux or storage in the cell, though iron content of ferritin could be increased. Our data demonstrate, for the first time, that AA status affects iron transport and the expression of genes related to iron metabolism in Caco-2 cells, although the changes observed are not sufficient to explain the alteration in iron transport. We hypothesise that the effect on Fe transfer is mediated through an increased movement across the cell layer, rather than transfer across the cell membranes. [ABSTRACT FROM AUTHOR]

During pregnancy, iron is transported from mother to fetus across the placenta. Iron is essential for many biological processes, including the transfer of oxygen in blood, but it can also be toxic. Elaborate and elegant mechanisms have evolved to make sure that the potential for oxidative damage is minimized. In this article, we examine how iron is transferred from the maternal liver to the placenta, taken up, and transferred to the fetal liver. We consider the molecular mechanisms and how they are regulated. In addition, we use data from previously published articles to examine how the processes are regulated and what adaptations can occur to ameliorate the consequences of iron deficiency-an all too common problem in pregnancy, even in the developed world. Finally, we examine some of the many questions that remain about the transfer process and consider how nutrients interact and what the consequences of these interactions may be for the mother and her developing infant. [ABSTRACT FROM AUTHOR]

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCE) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and Ill copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family "zyklopen." [ABSTRACT FROM AUTHOR]

Imbalances in nutrition during pregnancy can lead to long-, as well as short-term consequences, a phenomenon known as fetal programming. However, there is little information about when the fetus is most sensitive to its environment during gestation. We hypothesize that different fetal systems are most vulnerable to nutritional stress during periods of maximal growth and differentiation. We used iron (Fe) deficiency, which causes hypertension in the offspring, to test this hypothesis. We examined development between embryonic day (E) 10.5 and 12.5, when cardiovascular development is maximal, using whole embryo culture. Female rats were fed Fe-deficient or control diet for 4 wk before mating and up to E10.5. The embryos were cultured for 48 h in 95% rat serum collected from males fed either a control or Fe-deficient diet. Growth was impaired and heart size increased in embryos taken from Fe-deficient mothers and cultured in deficient serum compared with control embryos cultured in control serum. To test whether restoring normal Fe levels could reverse these effects, we cultured embryos from control and deficient dams in either control or deficient medium. The yolk sac circulation of embryos from dams fed either diet cultured in deficient medium was less developed, with a thinner and less branched network than that in all embryos cultured in control serum. The heart was enlarged in embryos of deficient dams cultured in deficient serum compared with the heart size of those cultured in control serum. Culturing embryos in control serum reversed these changes. We conclude, therefore, that this period of cardiovascular organogenesis is one of the sensitive windows during which optimal Fe status is critical for normal development. [ABSTRACT FROM AUTHOR]

The genes responsible for Wilson disease and Menkes syndrome have been cloned and identified as copper ATPases. These enzymes form part of a large family of transporters, the P-type ATPases. Although copper ATPases share strong structural similarities with these other pumps, comparatively little is known about their physiologic function. In this review, we examine data relating to the Wilson disease protein, ATP7B, in the liver. We present evidence suggesting that ATP7B is located intracellularly, together with data suggesting that, at least in part, ATP7B may also be found on the canalicular membrane. We also examine the form of copper that the transporter recognizes. We then review data on the Long-Evans Cinnamon rat, a model for Wilson disease, and discuss what effect the Wilson disease mutation has on copper transport. Finally, we conclude that, although we have made major advances in our understanding of copper metabolism in the liver, there are still many questions awaiting answers. [ABSTRACT FROM AUTHOR]

Investigates the effect of the micronutrients iron (Fe) and copper (Cu) on pregnancy outcome. Transfer of nutrients from mother to fetus across the placenta during pregnancy; Upregulation of the proteins involved in Fe transfer; Consequences of Fe deficiency during pregnancy; Increase of Cu levels in the maternal liver due to Fe deficiency.

Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA — strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency. [ABSTRACT FROM AUTHOR]

Iron deficiency during pregnancy causes problems both for the mother and fetus. Iron deficiency is known to have secondary effects on copper metabolism. In this study, we use a rat model to examine the effect of iron deficiency on copper levels in maternal and fetal tissue. We assess whether the effects of iron deficiency on copper metabolism are due to alterations in mRNA levels of proteins of copper transport. Rowett Hooded Lister rats were fed diets with four different iron contents before and during pregnancy. Maternal and fetal samples were collected on day 21 of gestation. Copper and iron levels of liver and placenta were analyzed, mRNA levels of genes involved in copper transport were studied, and copper oxidase activity measured. Reduced dietary iron was found to increase maternal liver copper, inversely correlating with iron levels. Correspondingly, copper and ceruloplasmin increased in maternal serum. The placenta showed the greatest increase in copper levels. As the iron content of the maternal diet decreased so did the iron and copper levels in the fetal liver. In all tissues examined, mRNA expression for CTR1, ATOX1, ATP7A, and ATP7B was unchanged by iron deficiency. However, copper oxidase activity in maternal serum and placenta was increased. Our study in a rat model demonstrates that iron deficiency during pregnancy has a differential effect on copper metabolism in the mother and fetus. It is clear from this study that the changes in copper levels that accompany iron deficiency are not mediated by changes in transcription of the genes involved in copper transport. [Copyright &y& Elsevier]

Iron deficiency impairs vitamin A (VA) metabolism in the rat but the mechanisms involved are unknown and the effect during development has not been investigated. We investigated the effect of pregnancy and maternal iron deficiency on VA metabolism in the mother and fetus. 54 rats were fed either a control or iron deficient diet for 2weeks prior to mating and throughout pregnancy. Another 15 female rats followed the same diet and were used as non-pregnant controls. Maternal liver, placenta and fetal liver were collected at d21 for total VA, retinol and retinyl ester (RE) measurement and VA metabolic gene expression analysis. Iron deficiency increased maternal hepatic RE (P<.05) and total VA (P<.0001), fetal liver RE (P<.05), and decreased placenta total VA (P<.05). Pregnancy increased Cellular Retinol Binding Protein (CRBP)-II gene expression by 7 fold (P=.001), decreased VA levels (P=.0004) and VA metabolic gene expression (P<.0001) in the liver. Iron deficiency increased hepatic CRBPII expression by a further 2 fold (P=.044) and RBP4 by~20% (P=.005), increased RBPR2 and decreased CRBPII, LRAT, and TTR in fetal liver, while it had no effect on VA metabolic gene expression in the placenta. Hepatic CRBPII expression is increased by pregnancy and further increased by iron deficiency, which may play an important role in VA metabolism and homeostasis. Maternal iron deficiency also alters VA metabolism in the fetus, which is likely to have consequences for development. [ABSTRACT FROM AUTHOR]

Abstract: This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using 125I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein – iron responsive element regulation mechanism. [Copyright &y& Elsevier]

Estrogen and progesterone regulate α, β, and γENaC subunit mRNA levels in female rat kidney. Background. Estrogen and progesterone regulate α, β, and γ amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA levels in female rat kidney. Renal Na+ handling differs between males and females. Further, within females Na+ metabolism changes during the menstrual cycle and pregnancy. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated transport of Na+ via the amiloride-sensitive Na+ channel. This study examines the role of the female gender steroids in the regulation of expression of ENaC. Methods. We measured ENaC subunit mRNA levels in rat kidney using Northern blotting. Kidneys were taken from male and females at different ages and from adult ovariectomized rats treated with 17-β-estradiol benzoate (estrogen) and/or progesterone for 8 or 24 hours. Results. The abundance of α, β, and γENaC mRNA was significantly higher in female compared to male rat kidneys from 10 weeks of age ( P= 0.001, P= 0.004, and P= 0.02, N= 10, respectively). These differences were abolished in ovariectomized rats. Treatment of ovariectomized rats with estrogen increased αENaC mRNA abundance in the kidney at both 8 and 24 hours ( P < 0.05, N= 6; and P < 0.05, N= 7, respectively). Progesterone inhibited the effect of estrogen on αENaC mRNA at 8 hours but when given alone increased γENaC mRNA ( P < 0.05, N= 3). Neither hormone, alone or in combination, had any significant effect on βENaC mRNA levels at 8 or 24 hours. Conclusion. Female gonadal steroids differentially modulate expression of ENaC subunit mRNA in the rat kidney. [ABSTRACT FROM AUTHOR]