Perspective: What Makes It So Difficult to Mitigate Worldwide Anemia Prevalence?

Anemia can be related to decreased production or increased loss of erythrocytes, or both, leading to many underlying and often overlapping causes. A largely cereal-based diet with plenty of phytates, polyphenols, and other ligands that inhibit intestinal iron absorption predominated in preindustrial Europe and predominates in present-day developing countries alike. In both situations, we find poor hygienic conditions, which frequently lead to anemia of inflammation. The large number of possible causes and their interaction shows why it is so difficult to mitigate anemia prevalence. Diagnostic biomarkers are required to differentiate the different types of anemia and to treat them appropriately. Some of them are well established in adults [e.g., concentrations of serum ferritin, soluble transferrin receptor (sTfR), and serum iron or the ratio of sTfR to log ferritin]. Others, such as serum hepcidin, hold considerable promise, although they are not yet widely used. A particular issue is to establish reference values for biomarkers in infants and children at different ages. The fact that resource-rich postindustrial societies have a very low prevalence of iron-deficiency anemia offers hope that common types of anemia can be eliminated. In contrast, inborn forms of anemia, such as thalassemia, and anemias related to underlying diseases (e.g., bleeding tumors or peptic ulcers, gynecologic blood losses, or renal diseases) require an operational health system to be addressed appropriately.

The Growth Attainment, Hematological, Iron Status and Inflammatory Profile of Guatemalan Juvenile End-Stage Renal Disease Patients.

BACKGROUND: Stunting, anemia and inflammation are frequently observed in children with end-stage renal disease (ESRD). OBJECTIVES: To assess anthropometric, hematological and inflammatory data and to study their potential interrelationship in Guatemalan juveniles undergoing hemodialysis (HD) and peritoneal dialysis (PD). METHODS: 54 juveniles 7-20 years of age were recruited in FUNDANIER, Guatemala City: 27 on HD and 27 PD. Hemoglobin, serum iron, transferrin, serum transferrin receptor (sTfR), serum ferritin, transferrin saturation and iron-binding capacity, white blood cell count (WBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), as well as IL-6, IL-1 and TNF-α, weight and height were determined by standard methods. Hepcidin-25 (Hep-25) was assessed by weak cation exchange time-of-flight mass-spectrometry. RESULTS: 92% and 55% of HD and PD children, respectively, were stunted and 95% and 85% were anemic. Among iron status biomarkers, serum ferritin was massively increased and significantly higher in the HD group compared to the PD group. Hep-25 was also greatly elevated in both groups. 41% of HD patients showed increments in three or more inflammatory biomarkers, while it was 2 or less in all PD subjects. CONCLUSIONS: The degree of stunting, the prevalence and severity of anemia in Guatemalan juvenile ESRD far exceed the national statistics for this low-income Central American country. Ferritin and Hep-25 concentrations were elevated, with the latter to an extraordinary magnitude. Additional biomarkers of inflammation not directly related to iron status were elevated as well. The role of both disease- and environment-related factors in combination best explains the magnitude of the biomarker abnormalities.

Hfe and Hjv exhibit overlapping functions for iron signaling to hepcidin.

UNLABELLED: Functional inactivation of HFE or hemojuvelin (HJV) is causatively linked to adult or juvenile hereditary hemochromatosis, respectively. Systemic iron overload results from inadequate expression of hepcidin, the iron regulatory hormone. While HJV regulates hepcidin by amplifying bone morphogenetic protein (BMP) signaling, the role of HFE in the hepcidin pathway remains incompletely understood. We investigated the pathophysiological implications of combined Hfe and Hjv ablation in mice. Isogenic Hfe (-)/(-) and Hjv (-)/(-) mice were crossed to generate double Hfe (-)/(-) Hjv (-)/(-) progeny. Wild-type control and mutant mice of all genotypes were analyzed for serum, hepatic, and splenic iron content, expression of iron metabolism proteins, and expression of hepcidin and Smad signaling in the liver, in response to a standard or an iron-enriched diet. As expected, Hfe (-)/(-) and Hjv (-)/(-) mice developed relatively mild or severe iron overload, respectively, which corresponded to the degree of hepcidin inhibition. The double Hfe (-)/(-) Hjv (-)/(-) mice exhibited an indistinguishable phenotype to single Hjv (-)/(-) counterparts with regard to suppression of hepcidin, serum and hepatic iron overload, splenic iron deficiency, tissue iron metabolism, and Smad signaling, under both dietary regimens. We conclude that the hemochromatotic phenotype caused by disruption of Hjv is not further aggravated by combined Hfe/Hjv deficiency. Our results provide genetic evidence that Hfe and Hjv operate in the same pathway for the regulation of hepcidin expression and iron metabolism. KEY MESSAGES: Combined disruption of Hfe and Hjv phenocopies single Hjv deficiency. Single Hjv(-)/(-) and double Hfe(-)/(-)Hjv(-)/(-) mice exhibit comparable iron overload. Hfe and Hjv regulate hepcidin via the same pathway.

Iron metabolism in obesity: how interaction between homoeostatic mechanisms can interfere with their original purpose. Part II: epidemiological and historic aspects of the iron/obesity interaction.

The change from a mainly vegetarian fare to meat consumption went along with brain growth and increased insulin resistance to improve brain's glucose supply. Meat consumption increased iron bioavailability and, thus, physical and mental fitness. The "predation-release-hypothesis" proposes that group coordination, arms and fire abolished the survival advantage of lean individuals from predation. The "thrifty gene-hypothesis", in contrast, proposes that surviving repeated episodes of starvation increased efficiency of food utilization in the offspring; they learned to utilize every available calorie. As a consequence of either mechanism, improved food security will increase prevalence of obesity along with that of its fatal consequences, such as diabetes, hypertension, heart diseases, and cancer. Thus, improved food security collides with the biologically evolved mechanisms to store excessive calories in preparation for a famine that never came. The crash between homoeostatic mechanisms and human intervention caused the presently observed pandemia of obesity and explains why it is so difficult to avoid, in spite of its well known and often fatal consequences.

Iron metabolism in obesity: how interaction between homoeostatic mechanisms can interfere with their original purpose. Part I: underlying homoeostatic mechanisms of energy storage and iron metabolisms and their interaction.

Adipose tissue plasticity mediated by inflammation is an important evolutionary achievement to survive seasonal climate changes. It permits to store excessive calories and to release them if required, using inflammatory cells to remove the debris. This process is regulated by a complex interaction of cytokines (TNF-α, IL-6), adipokines (adiponectin, apelin, liptin), adhesion molecules (ICAM-1, VCAM-1, E-selectin) and transcription factors (NF-κB, HIF-1α). Iron mediates electron transfer as an essential component of e.g. myeloperoxidase, hemoglobin, cytochrome C and ribonucleotide reductase. Conversely, unbound iron can catalyze oxidation of lipids, proteins, and DNA. To balance the essential with the potentially toxic function requires an efficient iron homoeostasis. This is mediated by hepcidin's interaction with the iron-exporter ferroportin, to adapt intestinal iron absorption and body iron-sequestration to changes in demand. In addition, the interaction of iron-responsive elements (IRE) and iron-responsive proteins (IRP), the IRE/IRP-mechanism, regulates cellular iron homoeostasis. Obesity-induced inflammation interacts with both these mechanisms and disturbs iron availability by impairing its absorption, and by sequestering it in the reticuloendothelial system. Both mechanisms lead to anemia and reduce physical fitness which, in a vicious cycle, can support the development of pathological obesity. Thus, interaction between these two sets of beneficial regulatory mechanisms can become detrimental in situations of ample calorie supply.

Can iron supplementation be reconciled with benefits and risks in areas hyperendemic for malaria?

Malaria is associated with about a million fatalities annually, largely among young children in zones of intense malarial transmission. The last thing needed would be measures that might increase the severity of clinical malaria. Thus, the finding in a field trial on Pemba Island, Tanzania, that routine oral iron supplementation produced adverse effects in iron-sufficient subjects had a ripple effect throughout the international public health community; it has effectively paralyzed efforts to redress iron-deficiency anemia in malaria-endemic regions. From a Hippocratic perspective, we consider the de facto moratorium on oral supplementation in such circumstance as a prudent interim measure. Public health programs to combat iron-deficiency anemia cannot be denied indefinitely to malaria-endemic populations, but the universal campaigns of iron provision cannot simply resume in the manner of the past. Contemporary biological and epidemiological understanding of the coevolution of humans and their pathogens should be able to provide guidance within the context of the essential and harmful aspects of iron. From these evolutionary standpoints, we identify a series of unresolved dilemmas. Toward a way forward, we highlight the pros and cons, as well as possible directions toward short-term strengthening, within three domains: tailored oral iron compounds, iron administration targeted only to iron-deficient individuals through screening, and prudent use of antimalarial prophylaxis. Although the tension between the essentiality of iron for humans and its role in pathogen virulence looms through every consideration, this recognition is a starting point toward the weighing of appropriate options balancing benefits and safety.

Differences in circulating non-transferrin-bound iron after oral administration of ferrous sulfate, sodium iron EDTA, or iron polymaltose in women with marginal iron stores.

BACKGROUND: The adverse interactions between iron supplements and malaria have driven the assessment of new therapeutic options for anemia prophylaxis in areas holoendemic for falciparum malaria. OBJECTIVE: To determine the responses of circulating non-transferrin-bound iron (NTBI) and plasma iron to three different oral iron compounds--ferrous sulfate, sodium iron ethylenediaminetetraacetate (NaFeEDTA), and iron polymaltose (IPM)--in women with marginal iron stores. METHODS: Serum samples from 10 Guatemalan women with marginal iron stores were collected every 90 minutes over a period of 270 minutes, after the individually randomized administration of 100 mg of iron from each of the three studied iron compounds or water alone. Serum iron concentration was quantified by the ferrozine method, and circulating NTBI concentration was determined with a fluorometric competitive binding assay. Kinetic responses and maximal cumulative changes in serum concentrations of iron and NTBI were compared between the four treatments. Comparison was made with data from the same protocol in iron-adequate men. RESULTS: The serum iron and NTBI responses to ferrous sulfate were significantly greater than those to water and the other two iron compounds. Serum iron responses to IPM did not differ from those to water alone. CONCLUSIONS: The administration of the two "slow-release" iron compounds, NaFeEDTA and IPM, resulted in a highly significant suppression of the appearance of NTBI in the circulation in the postsupplement period. These two bioavailable forms of iron supplement could represent a safe option for supplementation in malarial areas. The slope of the iron-NTBI relationship is steeper in men than in women.

Iron regulatory proteins control a mucosal block to intestinal iron absorption.

Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

Oral administration of ferrous sulfate, but not of iron polymaltose or sodium iron ethylenediaminetetraacetic acid (NaFeEDTA), results in a substantial increase of non-transferrin-bound iron in healthy iron-adequate men.

BACKGROUND: Oral iron supplementation with ferrous sulfate (FeSO4) at dosage levels suggested by the international guidelines poses a safety hazard to young children with malaria. Exposure to loosely bound iron in the circulation has been advanced as a potential factor. OBJECTIVE: To evaluate the kinetics of circulating concentrations of plasma iron and non-transferrin-bound iron (NTBI) in response to oral iron administration in healthy adults. METHODS: Plasma samples were collected at 90-minute intervals over a period of 270 minutes from 10 healthy Guatemalan men after oral administration of water or 100 mg of iron from each of three iron compounds: FeSO4, sodium iron ethylenediaminetetraacetic acid (NaFeEDTA), and iron polymaltose. The four tests were administered in an individually randomized sequence. Serum iron concentration was measured spectrophotometrically by the ferrozine method, and NTBI concentration was measured by a fluorometric competitive binding assay. The kinetic response and the maximal and cumulative changes in circulating concentrations of the biomarkers of interest were compared. RESULTS: Serum iron and NTBI responses to oral administration of FeSO4 were significantly greater than responses to plain water or the other two iron compounds. NTBI concentrations after NaFeEDTA or iron polymaltose ingestion were not different from those determined after water intake. CONCLUSIONS: Administration of two iron compounds of proven bioavailability, but with complex absorption characteristics, is associated with a negligible NTBI response, potentially mitigating the safety concerns associated with iron supplementation in malarial areas.

Targeted provision of oral iron: the evolution of a practical screening option.

Universal oral iron supplementation, undertaken according to 1998 WHO guidelines, produced adverse consequences among some children in malaria-endemic areas. Prompted by the Pemba trial, which revealed excessive hospitalizations and deaths, WHO advised that iron supplementation in such regions be accompanied by previous screening for iron deficiency. This agenda, however, poses issues of cost, benefit, acceptability, technical feasibility, and reliability of such screening. The cost of equipment and personnel is balanced against savings from iron supplements spared and treatment for morbidity averted. Costs aside, the most efficacious acceptable screening approach for avoiding hospitalization and deaths must be fielded. Screening before supplementation can be used to assess hematological, iron, and possible inflammatory status to differentiate the source of decreased hemoglobin concentration. Iron deficiency has often been inferred from hematological status markers. The need for extraction of blood, albeit capillary in origin, and high assay costs limit the use of validated methods in screening. Noninvasive methods, i.e., not requiring the extraction of blood, provide the most acceptable and potentially least expensive approach for determining hematological or iron status. Although a noninvasive technique for iron and inflammatory status would be the ideal, it is unattained. Field-friendly, skin-probe hemoglobin devices, derived from instruments for clinical settings, are being developed and tested for eventual rollout in malarial areas. Given a firm grounding for the theoretical requirements needed to advance the screening agenda, evaluation and monitoring of the performance of screening devices can proceed hand in hand.

Response of urinary biomarkers of systemic oxidation to oral iron supplementation in healthy men.

BACKGROUND: Urinary biomarkers are used in assessment of severe, clinical oxidative stress. Little is known, however, about their diagnostic value within the normative range. OBJECTIVE: To evaluate the response of urinary thiobarbituric acid reactive substances (TBARS) and 8-hydroxy-2-deoxyguanosine (8-OHdG) as indicators of systemic oxidation in response to short-term oral iron and antioxidant supplementation. METHODS: Five healthy adult men participated in the pilot study phase and 12 in the definitive intervention trial. For 7 days each, separated by 12-day washouts, the subjects received different treatment regimens, consisting of 120 mg of iron, 120 mg of iron in refined palm oil, and 120 mg of iron in palm oil combined with one of the two doses of Carotino Tocotrienol Carotene Mixed Concentrate (CTCMC). Creatinine-normalized urinary TBARS and 8-OHdG concentrations were quantified in samples taken from subjects with and without active supplementation. Temporal and correlative associations between TBARS and 8-OHdG were explored. RESULTS: Daily intake of supplemental iron failed to produce any increment in urinary excretion of TBARS or 8-OHdG. However, a significant within-individual correlation between the urinary biomarkers was observed (Spearman r = 0.697, p < .0001, n = 466). Both doses of CTCMC significantly lowered urinary excretion of both oxidation indicators. CONCLUSIONS: Despite the lack of effect of oral iron on the biomarkers of systemic oxidation, they show a strong and significant mutual association within the nonpathological range of oxidative stress in healthy male adults.

Validity and correspondence of non-invasively determined hemoglobin concentrations by two trans-cutaneous digital measuring devices.

Hemoglobin (Hb) concentration is the central diagnostic indicator for anemia, including nutritional anemia. The objective of this study was to compare the Hb values determined by two portable, non-invasive devices across a wide Hb spectrum against formal laboratory measurements, and with each other. Eighty Guatemalan adults (40 highland men, 40 lowland pregnant women) provided venous blood for formal Hb colorimetric determination. Hb was also registered sequentially on the Rad-87™ pulse CO-Oximeter with Rainbow Set technology (Masimo) and Haemospect® (MBR Optical Systems) by non-invasive skin-probe contact procedures as per manufacturers' instructions. Whole blood Hb concentrations ranged from 7.8 to 18.5 g/dL (mean, 12.9±2.3 g/dL and median, 13.3 g/dL). Corresponding descriptive statistics were: range, 9.6 to 16.2 g/dL; mean, 12.1±1.5 g/dL; and median, 11.9 g/L, respectively, with the Rad-87™ (nail bed). They were: range, 8.7 to 15.8 g/dL; mean, 12.7±1.8 g/dL; and median13.0 g/dL for the Hemospect® for forearm contact. They were: range, 9.1 to 17.5 g/dL; mean, 13.2±2.1 g/dL; and median, 13.4 g/dL for palm contact. The Pearson correlation coefficient of venous blood Hb with the former device's Hb values was r=0.59 (p<0.001), and r=0.94 (p<0.001) and r=0.90 (p<0.001) with those of the latter device at the palm and forearm, respectively. The inter-site Lin coefficient was r=0.84. Sensitivity and specificity were variable across devices, depending on Hb cut-off and measurement procedures. With Hb cut-off values of <12.0 g/dL for adult (non-pregnant women and <13.0 g/dL for adult men), the Haemospect device's performance here would provide adequate potential for screening purposes.

Equivalent effects on fecal reactive oxygen species generation with oral supplementation of three iron compounds: ferrous sulfate, sodium iron EDTA and iron polymaltose.

BACKGROUND: In any context of iron supplementation in the prenatal prophylaxis or therapeutic dosage range, a large amount will remain unabsorbed and pass through the intestinal tract into the colonic digesta possibly causing increased oxidation. AIM: To compare the generation of fecal reactive oxygen species (ROS) in situ after daily consumption of 100 mg of elemental iron in three frequently used forms of iron supplements. METHODS: Ten healthy, iron-repleted adult males were investigated before and during supplementation with three oral iron compounds: 100 mg of oral iron were given as ferrous sulfate, Na Fe-EDTA and iron polymaltose for 6 days to each subject in an individually stratified sequence. Stool samples were collected and analyzed for iron content and the in situ generation of fecal ROS. RESULTS: Significant increases in fecal ROS generation were observed during oral iron supplementation. No statistical differences were seen in either residual concentrations of non-heme iron in stool or the level of fecal ROS generation between the three Fe compounds. There was, however, a significant association between the iron concentration in the stool and ROS generation. CONCLUSION: In spite of the differences in their chemical characteristics, none of the three distinct iron complexes reduced oxidative stress in the intestinal lumen.

Impact of oral iron challenges on circulating non-transferrin-bound iron in healthy Guatemalan males.

INTRODUCTION: Oral iron as a supplement has been associated with adverse health consequences, especially in the context of young children with active malaria. A potential aggravating role of non-transferrin-bound iron (NTBI) has been proposed. MATERIAL AND METHODS: NTBI responses in both a fasting and post-oral iron dosing situation were related to serum iron concentration and ferritin status. Fasting and 1, 2, and 3 h postdose serum samples were obtained in conjunction with oral ferrous sulfate supplementation in aqueous solution of 0, 15, 30, 60, 120 and 240 mg Fe in a cohort of 8 healthy Guatemalan men over a 9-week metabolic protocol. Hemoglobin, serum ferritin, percent transferrin saturation, serum iron and NTBI were all measured. RESULTS: Circulating levels of serum iron and NTBI increased in a graded fashion in response to oral iron, with the relative increment for NTBI slightly greater than that of iron. Detectable NTBI was occasionally measured in fasting specimens, more frequently in subjects with high ferritin status. Post-iron NTBI responses, by contrast, were higher in normal-ferritin subjects in absolute terms, and rose with increasing postabsorptive serum iron responses. DISCUSSION: The appearance and response of circulating NTBI were consistent with recognized principles of iron regulation.

Absorption of iron from ferritin is independent of heme iron and ferrous salts in women and rat intestinal segments.

Ferritin iron from food is readily bioavailable to humans and has the potential for treating iron deficiency. Whether ferritin iron absorption is mechanistically different from iron absorption from small iron complexes/salts remains controversial. Here, we studied iron absorption (RBC (59)Fe) from radiolabeled ferritin iron (0.5 mg) in healthy women with or without non-ferritin iron competitors, ferrous sulfate, or hemoglobin. A 9-fold excess of non-ferritin iron competitor had no significant effect on ferritin iron absorption. Larger amounts of iron (50 mg and a 99-fold excess of either competitor) inhibited iron absorption. To measure transport rates of iron that was absorbed inside ferritin, rat intestinal segments ex vivo were perfused with radiolabeled ferritin and compared to perfusion with ferric nitrilotriacetic (Fe-NTA), a well-studied form of chelated iron. Intestinal transport of iron absorbed inside exogenous ferritin was 14.8% of the rate measured for iron absorbed from chelated iron. In the steady state, endogenous enterocyte ferritin contained >90% of the iron absorbed from Fe-NTA or ferritin. We found that ferritin is a slow release source of iron, readily available to humans or animals, based on RBC iron incorporation. Ferritin iron is absorbed by a different mechanism than iron salts/chelates or heme iron. Recognition of a second, nonheme iron absorption process, ferritin endocytosis, emphasizes the need for more mechanistic studies on ferritin iron absorption and highlights the potential of ferritin present in foods such as legumes to contribute to solutions for global iron deficiency.

Does lead use the intestinal absorptive pathways of iron? Impact of iron status on murine ²¹°Pb and 59Fe absorption in duodenum and ileum in vivo.

BACKGROUND: Human isotope studies and epidemiological trials are controversial as to whether lead absorption shares the absorptive pathways of iron and whether body lead content can be reduced by iron supplementation. AIM: To compare the impact of iron-deficiency on 59Fe- and ²¹°Pb-absorption rates in duodenal and ileal segments. METHODS: 59Fe- and ²¹°Pb-absorption was determined in ligated duodenal and ileal segments from juvenile and adult iron-deficient and iron-adequate C57Bl6 wild-type mice (n=6) in vivo at luminal concentrations corresponding to human exposure (Fe: 1 and 100 µmol/L; Pb: 1 µmol/L). RESULTS AND DISCUSSION: 59Fe-absorption increased 10-15-fold in iron-deficient duodena from adult and adolescent mice. Ileal 59Fe-absorption was 4-6 times lower than in iron-adequate duodena showing no adaptation to iron-deficiency. This in accordance to expectation as the divalent metal transport 1 (DMT1) shows low ileal expression levels. Juvenile 59Fe-absorption was about twice as high as in adult mice. In contrast, duodenal ²¹°Pb-absorption was increased only 1.5-1.8-fold in iron-deficiency in juvenile and adult mice and, again in contrast to 59Fe, ileal ²¹°Pb-absorption was as high as in iron-adequate duodena. CONCLUSIONS: The findings suggest a DMT1-independent pathway to mediate lead absorption along the entire small intestine in addition to DMT1-mediated duodenal uptake. Ileal lead absorption appears substantial, due the much longer residence of ingesta in the distal small intestine. Differences in lead-solubility and -binding to luminal ligands can, thus, explain the conflicting findings regarding the impact of iron-status on lead absorption. They need to be considered in future studies.