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.

Normative Fecal Calprotectin Concentrations in Guatemalan Preschoolers Are High Relative to Children Reported Elsewhere.

BACKGROUND: Calprotectin is a fecal marker of intraintestinal inflammation derived from activated enteric neutrophils and macrophages. It is useful as a clinical marker in inflammatory bowel diseases; furthermore, it may have a role in public health epidemiology. OBJECTIVES: The aim of the study was to describe the distribution of fecal calprotectin in Guatemalan preschool children sharing a common institutional diet; to relate it collectively to pediatric distributions in other geographic settings, and individually to concomitant indicators of intestinal infection or colonization and other descriptive features of the child. METHODS: Fecal samples were collected in 87 subjects, ages 2 to 7 years across 3 daycare centers sharing a common institutional menu, but from different ecological settings. Stools were examined, variously by routine light microscopy, quantitative egg counts, and a Giardia antigen test, for microbiological diagnosis, and an ELISA assay for fecal calprotectin (CalproLab). RESULTS: The median fecal calprotectin value was 58 mg/kg, with a mean of 98 ±â€Š136 mg/kg and a range from 10 to 950 mg/kg; 61% of values were above the manufacturer's cut-off for elevated concentration and 51% exceeded an age-adjusted criterion. There were no associations between sex, age, growth indicators, or fecal microbiological findings by microscopy or ELISA assays, alone or in combination. The central tendency (mean or median) and distribution were generally shifted to the right in relation to comparable reports from children across the world literature. CONCLUSIONS: Although specific, low-grade intestinal infections do not define calprotectin subgroups, right-shifted fecal calprotectin status in this population may reflect a general and diffuse stress of adverse environmental sanitation.

Strong Associations Exist among Oxidative Stress and Antioxidant Biomarkers in the Circulating, Cellular and Urinary Anatomical Compartments in Guatemalan Children from the Western Highlands.

BACKGROUND: A series of antioxidant enzymes and non-enzymatic compounds act to protect cells from uncontrolled propagation of free radicals. It is poorly understood, though, to what extent and how their interaction is harmonized. OBJECTIVES: To explore associative interactions among a battery of urinary and blood biomarkers of oxidative stress and enzymatic and non-enzymatic markers of the antioxidant defense system in children from low income households. METHODS: For this cross-sectional descriptive study, urine, red cells, and plasma were sampled in 82 preschool children attending three daycare centers in Quetzaltenango Guatemala. The urinary oxidative stress biomarkers studied were F2-isoprostanes and 8-hydroxy-deoxy-guanosine. Red cell enzyme activities measured were: catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Circulating non-enzymatic antioxidants selected were: retinol, tocopherols, ß-carotene and coenzymes Q9 and Q10. RESULTS: In a Spearman rank-order correlation hemi-matrix, of 55 paired combinations of the 11 biomarkers, 28 (51%) were significantly correlated among each other (p ≤ 0.05), with the strongest association being retinol and tocopherols (r = 0.697, p<0.001), and 4 associations (9%) showed a trend (p> 0.5 to ≤ 0.10). F2-isoprostanes showed the greatest number of cross-associations, having significant interactions with 8 of the 10 remaining biomarkers. Goodness-of-fit modeling improved or maintained the r value for 24 of the significant interactions and for one of the 5 borderline associations. Multiple regression backward stepwise analysis indicated that plasma retinol, ß-carotene and coenzyme Q10 were independent predictors of urinary F2-isoprostanes. CONCLUSION: Numerous significant associations resulted among biomarkers of oxidation and responders to oxidation. Interesting findings were the apparent patterns of harmonious interactions among the elements of the oxidation-antioxidation systems in this population.

Interaction of Giardia intestinalis and Systemic Oxidation in Preschool Children in the Western Highlands of Guatemala.

BACKGROUND: Guatemala is a country with the highest prevalence of stunting in under-5 children in the Americas, with a national average of 49.8%. Asymptomatic intestinal colonization with Giardia intestinalis is common in Guatemalan preschoolers and has been implicated as a factor in linear growth retardation. The potential mechanisms of any giardiasis-growth interaction have not been exhaustively explored. OBJECTIVES: The aim of the present study was to describe urine oxidative stress biomarkers and erythrocyte antioxidant enzyme activity, and to explore any association with prevalence or intensity of G intestinalis infection in preschoolers attending 3 government-subsidized day care centers in the Guatemalan Western Highlands. METHODS: Samples of feces, urine, and red blood cell (RBC) hemolysate were collected in a total of 74 preschoolers enrolled in 3 day care centers. Giardia prevalence and a proxy index for intensity were assessed by enzyme-linked immunosorbent assay (ELISA). Urinary biomarkers of oxidative damage to DNA (8-hydroxydeoxyguanosine [8-OHdG]) and to lipid (F2t 15-Isoprostane [F2-Iso]) were measured by ELISA. The erythrocyte activity of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), and glutathione peroxidase (GPX) were measured by respective spectroscopic substrate-based reaction assays. RESULTS: Median values of RBC CAT activity (P = 0.016) and urine F2-Iso (P = 0.023) differed between children who were positive (n = 39) and negative (n = 35) for Giardia. Similarly, G intestinalis intensity was significantly and positively associated with urinary F2-Iso (r = 0.446, P < 0.001), RBC SOD (r = 283, P = 0.014), and RBC CAT (r = 0.260, P = 0.025). CONCLUSION: The optical density reading of the fecal ELISA assay for G intestinalis has potential as a proxy for the intensity of infestation. In this respect, there exists an association of this intensity with indicators of the systemic oxidation.

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.

Variation in hydration status within the normative range is associated with urinary biomarkers of systemic oxidative stress in Guatemalan preschool children.

BACKGROUND: Researchers have increasingly sought noninvasive methods to determine health and nutritional status in humans. Easy and painless to collect, human urine is a source of noninvasive biomarkers. OBJECTIVE: We aimed to explore the relation between systemic oxidative stress biomarkers excreted in urine and urinary osmolality (Uosm). DESIGN: The current trial was a descriptive, cross-sectional study. We collected seventy-eight samples of 24-h urine in preschoolers who were attending daycare centers in the Western Highlands province of Quetzaltenango, Guatemala. After we measured the total urine volume (Uvol), the aliquot was stored for the later determination of Uosm as a hydration biomarker and to measure 15-isoprostane F2t (F2-Iso) and 8-hydroxydeoxyguanosine (8-OHdG) as biomarkers of cellular oxidation with the use of ELISA assay kits in Spain. Descriptive statistics and linear [Spearman rank-order (rs)] and nonlinear (goodness-of-fit) correlations were performed. RESULTS: Twenty-four hour Uvols ranged from 65 to 1670 mL, whereas the Uosm varied between 115 and 1102 mOsm/kg. With respect to oxidative biomarkers, the 24-h urinary output of F2-Iso and 8-OHdG had median values of 748 and 2793 ng/d, respectively. The Uvol correlated inversely and significantly with the concentrations of both oxidative biomarkers (F2-Iso rs = -0.603, P < 0.001; 8-OHdG rs = -0.433, P < 0.001), whereas the Uosm was correlated in a direct manner (F2-Iso rs = 0.541, P < 0.001; 8-OHdG rs = 0.782, P < 0.001) when analyzed as a concentration. Associations were weaker when they were analyzed as the total 24-h production. CONCLUSIONS: Preschool children from the Western Highlands of Guatemala show strong correlations between hydration status measured through the use of Uosm and biomarkers of oxidative stress in urine. Thus, a relatively superior hydration status is associated with a quantitative reduction in urinary excretion of systemic oxidation products. This trial was registered at clinicaltrials.gov as NCT02203890.

Associations among Inflammatory Biomarkers in the Circulating, Plasmatic, Salivary and Intraluminal Anatomical Compartments in Apparently Healthy Preschool Children from the Western Highlands of Guatemala.

BACKGROUND: Undernutrition and inflammation are related in many ways; for instance, non-hygienic environments are associated with both poor growth and immunostimulation in children. OBJECTIVE: To describe any existing interaction among different inflammation biomarkers measured in the distinct anatomical compartments of whole blood, feces, plasma and saliva. METHODS: In this descriptive, cross-sectional study, samples of whole blood, feces, plasma and saliva were collected on the 8th and last week of observation among 87 attendees (42 girls and 45 boys) of 3 daycare centers offering a common 40-day rotating menu in Guatemala's Western Highlands. Analyses included white blood cell count (WBC), fecal calprotectin, and plasmatic and salivary cytokines including IL-1B, IL-6, IL-8, IL-10 and TNF-α. Associations were assessed using Spearman rank-order and goodness-of-fit correlations, as indicated, followed by backwards-elimination multiple regression analyses to determine predictor variables for IL-10 in both anatomical compartments. RESULTS: Of a total of 66 cross-tabulations in the Spearman hemi-matrix, 22 (33%) were significantly associated. All 10 paired associations among the salivary cytokines had a significant r value, whereas 7 of 10 possible associations among plasma cytokines were significant. Associations across anatomical compartments, however, were rarely significant. IL-10 in both biological fluids were higher than corresponding reference values. When a multiple regression model was run in order to determine independent predictors for IL-10 in each anatomical compartment separately, IL-6, IL-8 and TNF-α emerged as predictors in plasma (r2 = 0.514) and IL-1B, IL-8 and TNF-α remained as independent predictors in saliva (r2 = 0.762). Significant cross-interactions were seen with WBC, but not with fecal calprotectin. CONCLUSION: Interactions ranged from robust within the same anatomical compartment to limited to nil across distinct anatomical compartments. The prominence of the anti-inflammatory cytokine, IL-10, in both plasma and saliva is consistent with its counter-regulatory role facing a broad front of elevated pro-inflammatory cytokines in the same compartment.

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.

59Fe-distribution in conditional ferritin-H-deleted mice.

The objective was to explore how ferritin-H deletion influences (59)Fe-distribution and excretion-kinetics in mice. Kinetics of (59)Fe-release from organs, whole-body excretion, and distribution-kinetics of intravenously injected (59)Fe trace amounts were compared in iron-deficient and iron-replete mice with (Fth(Δ/Δ)) and without (Fth(lox/lox)) conditional Mx-Cre-induced ferritin-H deletion. (59)Fe was released from spleen and liver beginning on day 2 and day 5 after ferritin-H deletion, respectively, but was not excreted from the body. Plasma-(59)Fe was cleared significantly faster in iron-deficient Fth(Δ/Δ)-mice than in iron-adequate Fth(lox/lox)-controls. (59)Fe-distribution showed a transient peak (e.g., in heart, kidney, muscle) in Fth(lox/lox) control mice, but not in ferritin-H-deleted Fth(Δ/Δ) mice 24 hours after (59)Fe injection. (59)Fe uptake into the liver and spleen was significantly lower in iron-deficient Fth(Δ/Δ) than in Fth(lox/lox) mice 24 hours and 7 days after injection, respectively, and rapidly appeared in circulating erythrocytes instead. The rate of (59)Fe release after ferritin-H deletion supports earlier data on ferritin turnover in mammals; released (59)Fe is not excreted from the body. Instead, (59)Fe is channeled into erythropoiesis and circulating erythrocytes significantly more extensively and faster. Along with a lack of transient interim (59)Fe storage (e.g., in the heart and kidney), this finding is evidence for ferritin-related iron storage-capacity affecting rate and extent of iron utilization.

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.