Iron Homeostasis During Pregnancy: Maternal, Placental, and Fetal Regulatory Mechanisms.

Pregnancy entails a large negative balance of iron, an essential micronutrient. During pregnancy, iron requirements increase substantially to support both maternal red blood cell expansion and the development of the placenta and fetus. As insufficient iron has long been linked to adverse pregnancy outcomes, universal iron supplementation is common practice before and during pregnancy. However, in high-resource countries with iron fortification of staple foods and increased red meat consumption, the effects of too much iron supplementation during pregnancy have become a concern because iron excess has also been linked to adverse pregnancy outcomes. In this review, we address physiologic iron homeostasis of the mother, placenta, and fetus and discuss perturbations in iron homeostasis that result in pathological pregnancy. As many mechanistic regulatory systems have been deduced from animal models, we also discuss the principles learned from these models and how these may apply to human pregnancy.

Multiple Indicators of Undernutrition, Infection, and Inflammation in Lactating Women Are Associated with Maternal Iron Status and Infant Anthropometry in Panama: The MINDI Cohort.

Maternal infections, nutrient deficiencies, and inflammation (MINDI) co-exist in lactating indigenous women in Panama, but their impact on maternal iron status and infant growth is unknown. For this secondary analysis of cross-sectional data of lactating mothers from our MINDI cohort, we investigated associations of MINDI variables with maternal anemia, elevated serum transferrin receptor (sTfR), low serum iron, hepcidin, ferritin, and infant weight-for-age (WAZ), length-for-age (LAZ), and head-circumference-for-age (HCAZ) Z-scores in 99 mother-infant dyads. A bootstrapping resampling procedure preselected covariates for inclusion in multivariable regressions models from chronic maternal infections and nutritional status [folate, vitamins A, D, retinol-binding protein (RBP), insulin-growth factor-1 (IGF-1)] and inflammation [C-reactive protein (CRP), cytokines, platelet indices] indicators. Anemia was prevalent (53.5%) but underestimated due to widespread low plasma volume (<2.2 L, 79.9%) and was associated with indicators of malnutrition [lower IGF-1, body mass index (BMI), vitamin D, and intake of green/leafy vegetables], but not inflammation. Higher CRP was associated with lower serum iron, and higher hepcidin and ferritin, whereas maternal platelets were associated with lower HCAZ (ß = −0.22), WAZ (ß = −0.17), and LAZ (ß = −0.17). Higher LAZ was also associated with maternal serum vitamin D (ß = 0.23), whereas maternal iron supplementation lowered LAZ (ß = −0.22). Assessment of iron status in this MINDI cohort is complex and supplementation strategies must consider consequences for both the mother and the infant.

Hepcidin and Erythroferrone Complement the Athlete Biological Passport in the Detection of Autologous Blood Transfusion.

PURPOSE: We investigated whether hepcidin and erythroferrone (ERFE) could complement the athlete biological passport (ABP) in indirectly detecting a 130-mL packed red blood cells (RBC) autologous blood transfusion. Endurance performance was evaluated. METHODS: Forty-eight healthy men ( n = 24) and women ( n = 24) participated. Baseline samples were collected weekly followed by randomization to a blood transfusion (BT, n = 24) or control group (CON, n = 24). Only the BT group donated 450 mL whole blood from which 130 mL red blood cell was reinfused 4 wk later. Blood samples were collected 3, 7, 14, 21, and 28 d after donation, and 3, 6, and 24 h and 2, 3, and 6 d after reinfusion. In the CON group samples were collected with the same frequency. Endurance performance was evaluated by a 650-kCal time trial ( n = 13) before and 1 and 6 d after reinfusion. RESULTS: A time-treatment effect existed ( P < 0.05) for hepcidin and ERFE. Hepcidin was increased ( P < 0.01) ~110 and 89% 6 and 24 h after reinfusion. Using an individual approach (99% specificity, e.g., allowing 1:100 false-positive), sensitivities, i.e., true positives, of 30% and 61% was found for hepcidin and ERFE, respectively. For the ABP, the most sensitive marker was Off-hr score ([Hb] (g·L -1 ) - 60 × âˆšRET%) ( P < 0.05) with a maximal sensitivity of ~58% and ~9% after donation and reinfusion, respectively. Combining the findings for hepcidin, ERFE, and the ABP yielded a sensitivity across all time-points of 83% after reinfusion in BT. Endurance performance increased 24 h (+6.4%, P < 0.01) and 6 d after reinfusion (+5.8%, P < 0.01). CONCLUSIONS: Hepcidin and ERFE may serve as biomarkers in an antidoping context after an ergogenic, small-volume blood transfusion.

Enteral ferric citrate absorption is dependent on the iron transport protein ferroportin.

Ferric citrate is approved as an iron replacement product in patients with non-dialysis chronic kidney disease and iron deficiency anemia. Ferric citrate-delivered iron is enterally absorbed, but the specific mechanisms involved have not been evaluated, including the possibilities of conventional, transcellular ferroportin-mediated absorption and/or citrate-mediated paracellular absorption. Here, we first demonstrate the efficacy of ferric citrate in high hepcidin models, including Tmprss6 knockout mice (characterized by iron-refractory iron deficiency anemia) with and without adenine diet-induced chronic kidney disease. Next, to assess whether or not enteral ferric citrate absorption is dependent on ferroportin, we evaluated the effects of ferric citrate in a tamoxifen-inducible, enterocyte-specific ferroportin knockout murine model (Villin-Cre-ERT2, Fpnflox/flox). In this model, ferroportin deletion was efficient, as tamoxifen injection induced a 4000-fold decrease in duodenum ferroportin mRNA expression, with undetectable ferroportin protein on Western blot of duodenal enterocytes, resulting in a severe iron deficiency anemia phenotype. In ferroportin-deficient mice, three weeks of 1% ferric citrate dietary supplementation, a dose that prevented iron deficiency in control mice, did not improve iron status or rescue the iron deficiency anemia phenotype. We repeated the conditional ferroportin knockout experiment in the setting of uremia, using an adenine nephropathy model, where three weeks of 1% ferric citrate dietary supplementation again failed to improve iron status or rescue the iron deficiency anemia phenotype. Thus, our data suggest that enteral ferric citrate absorption is dependent on conventional enterocyte iron transport by ferroportin and that, in these models, significant paracellular absorption does not occur.

Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy.

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.

INTERGROWTH-21 Identifies High Prevalence of Low Symphysis-Fundal Height in Indigenous Pregnant Women Experiencing Multiple Infections, Nutrient Deficiencies, and Inflammation: The Maternal Infections, Nutrient Deficiencies, and Inflammation (MINDI) Cohort.

BACKGROUND: In the absence of ultrasound, symphysis-fundal height (SFH) can assess maternal-fetal well-being as it is associated with gestational age, fetal weight, and amniotic fluid volume. However, other modifiers of SFH, including maternal infections, nutrient deficiencies, and inflammation (MINDI), have not been widely explored. OBJECTIVES: Our objectives were 2-fold: 1) to assess prevalence of low SFH in indigenous Panamanian women using both Pan-American Health Organization (PAHO) and INTERGROWTH-21 standards and 2) to explore associations of SFH with maternal health indicators: infections (oral, skin, urogenital, nematode infections), nutrient deficiencies [protein and iron indicators (ferritin, serum iron, serum transferrin receptor, hepcidin), folate, and vitamins A, D, and B-12], and inflammation [leukocytes, C-reactive protein (CRP), cytokines]. METHODS: For this cross-sectional study, low-SFH-for-gestational-age was assessed using PAHO and INTERGROWTH <10th centile in 174 women at ≥16 weeks of gestation. Bootstrapping selected MINDI variables for inclusion in multivariable fractional polynomial (MFP) logistic regressions for low SFH. Associations of MINDI variables with hepcidin were also investigated. RESULTS: Prevalence of low SFH was 8% using PAHO, but using INTERGROWTH, 50.6% had SFH <10th centile, including 37.9% <3rd centile. Both PAHO-SFH <10th centile and INTERGROWTH-SFH <3rd centile were associated with higher hepcidin (OR = 1.12, P = 0.008, and OR = 3.04, P = 0.001, respectively) and with lower TNF-α (OR = 0.73, P = 0.012, and OR = 0.93, P = 0.015, respectively). Wood-smoke exposure increased the odds of PAHO-SFH <10th centile (OR = 1.19, P = 0.009), whereas higher BMI decreased the odds of INTERGROWTH-SFH <3rd centile (OR = 0.87, P = 0.012). Lower pulse pressure (OR = 0.90, P = 0.009) and lower inflammatory responses [lower lymphocytes (OR = 0.21, P = 0.026), IL-17 (OR = 0.89, P = 0.011)] distinguished SFH <3rd centile from SFH ≥3rd to <10th centiles using INTERGROWTH-21 standards. The MFP regression for hepcidin controlling for SFH (adjusted R 2 = 0.40, P = 0.001) revealed associations with indicators of inflammation (CRP, P < 0.0001; IL-17, P = 0.012), acidic urinary pH (P = 0.008), and higher intake of supplements (P = 0.035). CONCLUSIONS: Associations of low SFH with MINDI variables, including hepcidin, highlight its potential for early detection of multicausal in utero growth faltering.

Effects of maternal iron status on placental and fetal iron homeostasis.

Iron deficiency is common worldwide and is associated with adverse pregnancy outcomes. The increasing prevalence of indiscriminate iron supplementation during pregnancy also raises concerns about the potential adverse effects of iron excess. We examined how maternal iron status affects the delivery of iron to the placenta and fetus. Using mouse models, we documented maternal homeostatic mechanisms that protect the placenta and fetus from maternal iron excess. We determined that under physiological conditions or in iron deficiency, fetal and placental hepcidin did not regulate fetal iron endowment. With maternal iron deficiency, critical transporters mediating placental iron uptake (transferrin receptor 1 [TFR1]) and export (ferroportin [FPN]) were strongly regulated. In mice, not only was TFR1 increased, but FPN was surprisingly decreased to preserve placental iron in the face of fetal iron deficiency. In human placentas from pregnancies with mild iron deficiency, TFR1 was increased, but there was no change in FPN. However, induction of more severe iron deficiency in human trophoblast in vitro resulted in the regulation of both TFR1 and FPN, similar to what was observed in the mouse model. This placental adaptation that prioritizes placental iron is mediated by iron regulatory protein 1 (IRP1) and is important for the maintenance of mitochondrial respiration, thus ultimately protecting the fetus from the potentially dire consequences of generalized placental dysfunction.

Single versus Split Dose of Iron Optimizes Hemoglobin Mass Gains at 2106 m Altitude.

PURPOSE: To determine if a single versus a split equivalent daily dose of elemental iron was superior for hemoglobin mass (Hbmass) gains at altitude while minimizing gastrointestinal (GI) discomfort. METHODS: Twenty-four elite runners attended a 3.1 ± 0.3 wk training camp (Flagstaff, AZ; 2106 m). A two-group design, randomized and stratified to baseline Hbmass, sex, and ferritin (>30 µ·L), was implemented daily as: 1) single dose of 1 × 200 mg (PM only, SINGLE) versus 2) split dose of 2 × 100 mg (AM and PM; SPLIT) elemental iron (ferrous fumarate). The Hbmass and venipuncture assessments were completed upon arrival and departure (±2 d) from camp for ferritin, hepcidin, and erythroferrone (ERFE) concentrations. Validated food frequency, GI distress, menstrual blood loss (MBL) and training questionnaires were implemented throughout. Univariate analysis was used to compare Hbmass, with baseline ferritin, dietary iron intake, MBL, and training volume used as covariates. RESULTS: Both conditions increased Hbmass from baseline (P < 0.05), with SINGLE (867.3 ± 47.9 g) significantly higher than SPLIT (828.9 ± 48.9 g) (P = 0.048). The GI scores were worse in SINGLE for weeks 1 and 2 combined (SINGLE, 18.0 ± 6.7 points; SPLIT, 11.3 ± 6.9 points; P = 0.025); however, GI scores improved by week 3, resulting in no between-group differences (P = 0.335). Hepcidin significantly decreased over time (P = 0.043) in SINGLE, with a nonsignificant decrease evident in SPLIT (~22%). ERFE significantly decreased in both groups (~28.5%; P < 0.05). No between-group differences existed for ERFE, hepcidin, food frequency, MBL, or daily training outcomes (P > 0.05). CONCLUSIONS: A single nightly 200-mg dose of elemental iron was superior to a split dose for optimizing Hbmass changes at altitude in runners over an approximately 3-wk training camp.

Intravenous Iron Does Not Augment the Hemoglobin Mass Response to Simulated Hypoxia.

PURPOSE: Iron is integral for erythropoietic adaptation to hypoxia, yet the importance of supplementary iron compared with existing stores is poorly understood. The aim of the present study was to compare the magnitude of the hemoglobin mass (Hbmass) in response to altitude in athletes with intravenous (IV), oral, or placebo iron supplementation. METHODS: Thirty-four, nonanemic, endurance-trained athletes completed 3 wk of simulated altitude (3000 m, 14 h·d), receiving two to three bolus iron injections (ferric carboxymaltose), daily oral iron supplementation (ferrous sulfate), or a placebo, commencing 2 wk before and throughout altitude exposure. Hbmass and markers of iron regulation were assessed at baseline (day -14), immediately before (day 0), weekly during (days 8 and 15), and immediately, 1, 3, and 6 wk after (days 22, 28, 42, and 63) the completion of altitude exposure. RESULTS: Hbmass significantly increased after altitude exposure in athletes with IV (mean % [90% confidence interval (CI)], 3.7% [2.8-4.7]) and oral (3.2% [2.2-4.2]) supplementation and remained elevated at 7 d postaltitude in oral (2.9% [1.5-4.3]) and 21 d after in IV (3.0% [1.5-4.6]) supplementation. Hbmass was not significantly higher than baseline at any time point in placebo. CONCLUSIONS: Iron supplementation appears necessary for optimal erythropoietic adaptation to altitude exposure. IV iron supplementation during 3 wk of simulated live high-train low altitude training offered no additional benefit in terms of the magnitude of the erythropoietic response for nonanemic endurance athletes compared with oral supplementation.

Therapeutic recommendations in HFE hemochromatosis for p.Cys282Tyr (C282Y/C282Y) homozygous genotype.

Although guidelines are available for hereditary hemochromatosis, a high percentage of the recommendations within them are not shared between the different guidelines. Our main aim is to provide an objective, simple, brief, and practical set of recommendations about therapeutic aspects of HFE hemochromatosis for p.Cys282Tyr (C282Y/C282Y) homozygous genotype, based on the published scientific studies and guidelines, in a form that is reasonably comprehensible to patients and people without medical training. This final version was approved at the Hemochromatosis International meeting on 12th May 2017 in Los Angeles.

Iron homeostasis during pregnancy.

During pregnancy, iron needs to increase substantially to support fetoplacental development and maternal adaptation to pregnancy. To meet these iron requirements, both dietary iron absorption and the mobilization of iron from stores increase, a mechanism that is in large part dependent on the iron-regulatory hormone hepcidin. In healthy human pregnancies, maternal hepcidin concentrations are suppressed in the second and third trimesters, thereby facilitating an increased supply of iron into the circulation. The mechanism of maternal hepcidin suppression in pregnancy is unknown, but hepcidin regulation by the known stimuli (i.e., iron, erythropoietic activity, and inflammation) appears to be preserved during pregnancy. Inappropriately increased maternal hepcidin during pregnancy can compromise the iron availability for placental transfer and impair the efficacy of iron supplementation. The role of fetal hepcidin in the regulation of placental iron transfer still remains to be characterized. This review summarizes the current understanding and addresses the gaps in knowledge about gestational changes in hematologic and iron variables and regulatory aspects of maternal, fetal, and placental iron homeostasis.

Hepcidin-mediated iron sequestration protects against bacterial dissemination during pneumonia.

Gram-negative pneumonia is a dangerous illness, and bacterial dissemination to the bloodstream during the infection is strongly associated with death. Antibiotic resistance among the causative pathogens has resulted in diminishing treatment options against this infection. Hepcidin is the master regulator of extracellular iron availability in vertebrates, but its role in the context of host defense is undefined. We hypothesized that hepcidin-mediated depletion of extracellular iron during Gram-negative pneumonia protects the host by limiting dissemination of bacteria to the bloodstream. During experimental pneumonia, hepcidin was induced in the liver in an IL-6-dependent manner and mediated a rapid decline in plasma iron. In contrast, hepcidin-deficient mice developed a paradoxical increase in plasma iron during infection associated with profound susceptibility to bacteremia. Incubation of bacteria with iron-supplemented plasma enhanced bacterial growth in vitro, and systemic administration of iron to WT mice similarly promoted increased susceptibility to bloodstream infection. Finally, treatment with a hepcidin analogue restored hypoferremia in hepcidin-deficient hosts, mediated bacterial control, and improved outcomes. These data show hepcidin induction during pneumonia to be essential to preventing bacterial dissemination by limiting extracellular iron availability. Hepcidin agonists may represent an effective therapy for Gram-negative infections in patients with impaired hepcidin production or signaling.

Hepcidin in the diagnosis of iron disorders.

The discovery of the iron-regulatory hormone hepcidin in 2001 has revolutionized our understanding of iron disorders, and its measurement should advance diagnosis/treatment of these conditions. Although several assays have been developed, a gold standard is still lacking, and efforts toward harmonization are ongoing. Nevertheless, promising applications can already be glimpsed, ranging from the use of hepcidin levels for diagnosing iron-refractory iron deficiency anemia to global health applications such as guiding safe iron supplementation in developing countries with high infection burden.

Systemic and tumor level iron regulation in men with colorectal cancer: a case control study.

BACKGROUND: Increased cellular iron exposure is associated with colorectal cancer (CRC) risk. Hepcidin, a liver peptide hormone, acts as the primary regulator of systemic iron status by blocking iron release from enterocytes into plasma. Concentrations are decreased during low iron status and increased during inflammation. The role of hepcidin and the factors influencing its regulation in CRC remains largely unknown. This study explored systemic and tumor level iron regulation in men with CRC. METHODS: The participants were 20 CRC cases and 20 healthy control subjects. Colonic tissue (adenocarcinoma [cases] healthy mucosa [controls]) was subjected to quantitative PCR (hepcidin, iron transporters and IL-6) and Perls' iron staining. Serum was analyzed using ELISA for hepcidin, iron status (sTfR) and inflammatory markers (CRP, IL-6, TNF-α). Anthropometrics, dietary iron intake and medical history were obtained. RESULTS: Cases and controls were similar in demographics, medication use and dietary iron intake. Systemically, cases compared to controls had lower iron status (sTfR: 21.6 vs 11.8 nmol/L, p < 0.05) and higher marker of inflammation (CRP: 8.3 vs 3.4 µg/mL, p < 0.05). Serum hepcidin was mildly decreased in cases compared to controls; however, it was within the normal range for both groups. Within colonic tissue, 30% of cases (6/20) presented iron accumulation compared to 5% of controls (1/20) (χ(2) = 5.0; p < 0.05) and higher marker of inflammation (IL-6: 9.4-fold higher compared to controls, p < 0.05). Presence of adenocarcinoma iron accumulation was associated with higher serum hepcidin (iron accumulation group 80.8 vs iron absence group 22.0 ng/mL, p < 0.05). CONCLUSIONS: While CRC subjects had serum hepcidin concentrations in the normal range, it was higher given their degree of iron restriction. Inappropriately elevated serum hepcidin may reduce duodenal iron absorption and further increase colonic adenocarcinoma iron exposure. Future clinical studies need to assess the appropriateness of dietary iron intake or iron supplementation in patients with CRC.

Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload.

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as ß-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and ß-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7­9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.

Understanding the structure/activity relationships of the iron regulatory peptide hepcidin.

The peptide hormone hepcidin is a key homeostatic regulator of iron metabolism and involved in pathological regulation of iron in response to infection, inflammation, hypoxia, and anemia. It acts by binding to the iron exporter ferroportin, causing it to be internalized and degraded; however, little is known about the structure/activity relationships of the interaction of hepcidin with ferroportin. We show that there are key residues in the N-terminal region of hepcidin that influence its interaction with ferroportin, and we explore the structure/function relationships at these positions. A series of hepcidin mutants in which disulfide bonds were replaced with diselenide bonds showed no change in activity compared to native hepcidin. These results identify important constraints for the development of hepcidin congeners for the treatment of hereditary iron overload.

Liver iron concentrations and urinary hepcidin in beta-thalassemia.

BACKGROUND AND OBJECTIVES: Patients with beta-thalassemia, like those with genetic hemochromatosis, develop iron overload due to increased iron absorption, and their iron burden is further exacerbated by transfusion therapy. Hepcidin, a hepatic hormone, regulates systemic iron homeostasis by inhibiting the absorption of iron from the diet and the recycling of iron by macrophages. In turn, hepcidin release is increased by iron loading and inhibited by erythropoietic activity. Hepcidin deficiency is the cause of iron overload in most forms of hereditary hemochromatosis. We sought to determine hepcidin's role in the pathogenesis of iron overload in b-thalassemia. DESIGN AND METHODS: We assessed the degree of iron overload in thalassemia intermedia and major patients by measuring hepatic iron concentration in liver biopsy samples and serum ferritin, estimated erythropoietic drive by assaying soluble transferrin receptor and serum erythropoietin levels and correlated these with urinary hepcidin measurements. RESULTS: Urinary hepcidin levels in beta-thalassemia demonstrate severe hepcidin deficiency in thalassemia intermedia. There was a strong inverse relationship between urinary hepcidin levels and both erythropoietin and soluble transferrin receptor, markers of erythropoietic activity. In contrast, hepcidin levels were elevated in thalassemia major, presumably due to transfusions that reduce erythropoietic drive and deliver a large iron load. Despite similar liver iron concentrations in the two conditions, serum ferritin was much lower in thalassemia intermedia. INTERPRETATION AND CONCLUSIONS: In thalassemia intermedia, high erythropoietic drive causes severe hepcidin deficiency. The lack of hepcidin results in hyperabsorption of dietary iron, but also in iron depletion of macrophages, lowering their secretion of ferritin and, consequently, serum ferritin levels. In contrast, in thalassemia major, transfusions decrease erythropoietic drive and increase the iron load, resulting in relatively higher hepcidin levels. In the presence of higher hepcidin levels, dietary iron absorption is moderated and macrophages retain iron, contributing to higher serum ferritin. In the future, hepcidin measurements may allow a more accurate assessment of the degree of iron overload and the maldistribution of iron in thalassemia.