Erwin T. Wiegerinck

Hepcidin in obese children as a potential mediator of the association between obesity and iron deficiency.

CONTEXT Obesity and iron deficiency are two of the most common nutritional disorders worldwide. Several studies found higher rates of iron deficiency in obese than in normal-weight children. Hepcidin represents the main inhibitor of intestinal iron absorption, and its expression is increased in adipose tissue of obese patients. Leptin is able, in vitro, to raise hepcidin expression. OBJECTIVES Aims of this work were 1) to assess the association between poor iron status and obesity, 2) to investigate whether iron homeostasis of obese children may be modulated by serum hepcidin variations, and 3) to assess the potential correlation between leptin and serum hepcidin variations. METHODS Iron status and absorption as well as hepcidin, leptin, and IL-6 levels were studied in 60 obese children and in 50 controls. RESULTS Obese children showed lower iron and transferrin saturation (both P < 0.05) and higher hepcidin levels (P = 0.004) compared with controls. A direct correlation between hepcidin and obesity degree (P = 0.0015), and inverse correlations between hepcidin and iron (P = 0.04), hepcidin and transferrin saturation (P = 0.005), and hepcidin and iron absorption (P = 0.003) were observed. A correlation between leptin and hepcidin (P = 0.006) has been found. The correlation remained significant when adjusted for body mass index, sex, pubertal stage, and IL-6 values. CONCLUSIONS We propose that in obese patients, increased hepcidin production, at least partly leptin mediated, represents the missing link between obesity and disrupted iron metabolism.

Afebrile Plasmodium falciparum parasitemia decreases absorption of fortification iron but does not affect systemic iron utilization: a double stable-isotope study in young Beninese women

BACKGROUND Iron deficiency anemia (IDA) affects many young women in sub-Saharan Africa. Its etiology is multifactorial, but the major cause is low dietary iron bioavailability exacerbated by parasitic infections such as malaria. OBJECTIVE We investigated whether asymptomatic Plasmodium falciparum parasitemia in Beninese women would impair absorption of dietary iron or utilization of circulating iron. DESIGN Iron absorption and utilization from an iron-fortified sorghum-based meal were estimated by using oral and intravenous isotope labels in 23 afebrile women with a positive malaria smear (asexual P. falciparum parasitemia; > 500 parasites/μL blood). The women were studied while infected, treated, and then restudied 10 d after treatment. Iron status, hepcidin, and inflammation indexes were measured before and after treatment. RESULTS Treatment reduced low-grade inflammation, as reflected by decreases in serum ferritin, C-reactive protein, interleukin-6, interleukin-8, and interleukin-10 (P < 0.05); this was accompanied by a reduction in median serum hepcidin of ≈ 50%, from 2.7 to 1.4 nmol/L (P < 0.005). Treatment decreased serum erythropoietin and growth differentiation factor 15 (P < 0.05). Clearance of parasitemia increased geometric mean dietary iron absorption (from 10.2% to 17.6%; P = 0.008) but did not affect systemic iron utilization (85.0% compared with 83.1%; NS). CONCLUSIONS Dietary iron absorption is reduced by ≈ 40% in asymptomatic P. falciparum parasitemia, likely because of low-grade inflammation and its modulation of circulating hepcidin. Because asymptomatic parasitemia has a protracted course and is very common in malarial areas, this effect may contribute to IDA and blunt the efficacy of iron supplementation and fortification programs. This trial was registered at clinicaltrials.gov as NCT01108939.

Training Surface and Intensity : Inflammation, Hemolysis, and Hepcidin Expression

PURPOSE This investigation assessed the effects of training intensity and ground surface type on hemolysis, inflammation, and hepcidin activity during running. METHODS Ten highly trained male endurance athletes completed a graded exercise test, two continuous 10-km runs on a grass (GRASS) and a bitumen road surface (ROAD) at 75%-80% peak VO2 running velocity, and a 10 x 1-km interval running session (INT) at 90%-95% of the peak VO2 running velocity. Venous blood and urine samples were collected before, immediately after, and at 3 and 24 h after exercise. Serum samples were analyzed for circulating levels of IL-6, free hemoglobin (Hb), haptoglobin (Hp), iron, and ferritin. Urine samples were analyzed for changes in hepcidin expression. RESULTS After running, the IL-6 and free Hb were significantly greater, and serum Hp was significantly lower than preexercise values in all three conditions (P < 0.05). Furthermore, IL-6 levels and the change in free Hb from baseline were significantly greater in the INT compared with those in the GRASS (P < 0.05). There were no differences between the GRASS and ROAD training surfaces (P > 0.05). Serum iron and ferritin were significantly increased after exercise in all three conditions (P < 0.05) but were not different between trials. CONCLUSION Greater running intensities incur more inflammation and hemolysis, but these variables were not affected by the surface type trained upon.

Effects of Blood-Processing Protocols on Cell-free DNA Quantification in Plasma

Recently, qualitative analysis of cell-free DNA in blood plasma has attracted much interest for the diagnosis of cancer, fetal gender, Rhesus D status, and inherited disorders (1)(2). Other studies have shown that quantification of total plasma DNA may indicate fetal chromosomal aneuploidies and pregnancy-associated complications or the presence/recurrence of cancer (1)(3)(4). Irrespective of the type of study (qualitative or quantitative DNA analysis), it is important that cell-free plasma DNA is not contaminated with cellular DNA that interferes with analysis and accurate quantification. To prevent cellular DNA contamination, Chiu et al. (5) emphasized the need of preanalytical standardization of blood-processing protocols. The authors note that after centrifugation of blood at a low speed (800g), the amount of isolated DNA from plasma is affected by the presence of cells that remain in the plasma fraction. An additional centrifugation step (16 000g) or filtering of the plasma is necessary to produce absolutely cell-free plasma DNA. Because most laboratories, including ours, centrifuge blood at relatively low speeds (800–1500g) and store plasma without additional treatment, these results may have a serious impact on the usefulness of previously collected plasma samples for retrospective DNA analysis/quantification. To confirm the results of Chiu et al. (5), we collected EDTA blood in 7-mL Vacutainer Tubes from 18 healthy …

Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25+40 as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.

Hepcidin-25 is related to cardiovascular events in chronic haemodialysis patients

BACKGROUND The development of atherosclerosis may be enhanced by iron accumulation in macrophages. Hepcidin-25 is a key regulator of iron homeostasis, which downregulates the cellular iron exporter ferroportin. In haemodialysis (HD) patients, hepcidin-25 levels are increased. Therefore, it is conceivable that hepcidin-25 is associated with all-cause mortality and/or fatal and non-fatal cardiovascular (CV) events in this patient group. The aim of the current analysis was to study the relationship between hepcidin-25 and all-cause mortality and both fatal and non-fatal CV events in chronic HD patients. METHODS Data from 405 chronic HD patients included in the CONvective TRAnsport STudy (NCT00205556) were studied (62% men, age 63.7 ± 13.9 years [mean ± SD]). The median (range) follow-up was 3.0 (0.8-6.6) years. Hepcidin-25 was measured with mass spectrometry. The relationship between hepcidin-25 and all-cause mortality or fatal and non-fatal CV events was investigated with multivariate Cox proportional hazard models. RESULTS Median (interquartile range) hepcidin-25 level was 13.8 (6.6-22.5) nmol/L. During follow-up, 158 (39%) patients died from any cause and 131 (32%) had a CV event. Hepcidin-25 was associated with all-cause mortality in an unadjusted model [hazard ratio (HR) 1.14 per 10 nmol/L, 95% CI 1.03-1.26; P = 0.01], but not after adjustment for all confounders including high-sensitive C-reactive protein (HR 1.02 per 10 nmol/L, 95% CI 0.87-1.20; P = 0.80). At the same time, hepcidin-25 was significantly related to fatal and non-fatal CV events in a fully adjusted model (HR 1.24 per 10 nmol/L, 95% CI 1.05-1.46, P = 0.01). CONCLUSION Hepcidin-25 was associated with fatal and non-fatal CV events, even after adjustment for inflammation. Furthermore, inflammation appears to be a significant confounder in the relation between hepcidin-25 and all-cause mortality. These findings suggest that hepcidin-25 might be a novel determinant of CV disease in chronic HD patients.

The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study

Abstract Background: Iron deficiency is a commonly encountered problem in pregnancy and a frequently observed cause of pregnancy-associated anemia. We longitudinally assessed the iron regulatory hormone hepcidin during gestation and postpartum and related hepcidin to conventional indicators of iron status and inflammation. Methods: Thirty-one healthy pregnant women were included and 81 blood samples from the three trimesters, directly and 6 weeks postpartum were analyzed for hemoglobin, the iron parameters: iron, total iron binding capacity, transferrin saturation, ferritin, soluble transferrin receptor and hepcidin, and CRP and leucocytes as markers of inflammation. Results: Hepcidin concentration decreased gradually from the first to the second and third trimester to undetectable levels (≤0.5 nmol/L) which was paralleled by decreasing hemoglobin levels and changes in iron parameters indicative for iron deficiency. During gestation hepcidin levels correlated with iron parameters, but not with inflammatory markers. Postpartum, hepcidin increased immediately to levels similar as assessed at early pregnancy. Conclusions: We conclude that hepcidin levels were suppressed during the second and third trimester of pregnancy, which was likely determined by the occurrence of iron deficiency. These data give insight in iron homeostasis during normal pregnancy.

Hepcidin-25 in Chronic Hemodialysis Patients Is Related to Residual Kidney Function and Not to Treatment with Erythropoiesis Stimulating Agents

Hepcidin-25, the bioactive form of hepcidin, is a key regulator of iron homeostasis as it induces internalization and degradation of ferroportin, a cellular iron exporter on enterocytes, macrophages and hepatocytes. Hepcidin levels are increased in chronic hemodialysis (HD) patients, but as of yet, limited information on factors associated with hepcidin-25 in these patients is available. In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients. Baseline data from 405 patients (62% male; age 63.7±13.9 [mean SD]) enrolled in the CONvective TRAnsport STudy (CONTRAST; NCT00205556) were studied. Predialysis hepcidin concentrations were measured centrally with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patient-, laboratory- and treatment related characteristics were entered in a backward multivariable linear regression model. Hepcidin-25 levels were independently and positively associated with ferritin (p<0.001), hsCRP (p<0.001) and the presence of diabetes (p = 0.02) and inversely with the estimated glomerular filtration rate (p = 0.01), absolute reticulocyte count (p = 0.02) and soluble transferrin receptor (p<0.001). Men had lower hepcidin-25 levels as compared to women (p = 0.03). Hepcidin-25 was not associated with the maintenance dose of erythropoiesis stimulating agents (ESA) or iron therapy. In conclusion, in the currently studied cohort of chronic HD patients, hepcidin-25 was a marker for iron stores and erythropoiesis and was associated with inflammation. Furthermore, hepcidin-25 levels were influenced by residual kidney function. Hepcidin-25 did not reflect ESA or iron dose in chronic stable HD patients on maintenance therapy. These results suggest that hepcidin is involved in the pathophysiological pathway of renal anemia and iron availability in these patients, but challenges its function as a clinical parameter for ESA resistance.

Plasma hepcidin levels and anemia in old age. The Leiden 85-plus Study

Hepcidin, an important regulator of iron homeostasis, is suggested to be causally related to anemia of inflammation. The aim of this study was to explore the role of plasma hepcidin in anemia among older persons from the general population. The Leiden 85-Plus Study is a population-based study of 85-year olds in Leiden, the Netherlands. Eighty-five-year old inhabitants of Leiden were enrolled between September 1997 and September 1999. At the age of 86, plasma hepcidin was determined with time of flight mass spectrometry in 490 participants [160 (32.7%) male, 114 (23.3%) with anemia]. Anemia was defined according to criteria of the World Health Organization (hemoglobin level <13 g/dL for men and hemoglobin <12 g/dL for women). The median plasma hepcidin level was 3.0 nM [interquartile range (IQR) 1.8–4.9]. We found strong correlations between plasma hepcidin and body iron status, C-reactive protein and erythropoietin levels. Significantly higher hepcidin levels were found in participants with anemia of inflammation (P<0.01), in participants with anemia of kidney disease (P=0.01), and in participants with unexplained anemia (P=0.01) than in participants without anemia. Participants with iron-deficiency anemia had significantly lower plasma hepcidin levels than participants without anemia (P<0.01). In conclusion, older persons with anemia of inflammation have higher hepcidin levels than their counterparts without anemia. The potential clinical value of hepcidin in future diagnostic algorithms for anemia has to be explored.

Diurnal Rhythm Rather Than Dietary Iron Mediates Daily Hepcidin Variations

BACKGROUND The iron-regulating hormone hepcidin is a promising biomarker in the diagnosis of iron disorders. Concentrations of hepcidin have been shown to increase during the day in individuals who are following a regular diet. It is currently unknown whether these increases are determined by an innate rhythm or by other factors. We aimed to assess the effect of dietary iron on hepcidin concentrations during the day. METHODS Within a 7-day interval, 32 volunteers received an iron-deficient diet on 1 day and the same diet supplemented with 65 mg ferrous fumarate at 0815 and 1145 on another day. Blood was drawn to assess ferritin, hepcidin-25, and transferrin saturation (TS) throughout both days at 4 time points between 0800 (fasted) and 1600. A linear mixed model for repeated data was used to analyze the effect of iron intake on TS and hepcidin concentrations. RESULTS Baseline values of hepcidin at 0800 correlated significantly with ferritin (r = 0.61). During the day of an iron-deficient diet the mean TS was similar both in men and in women, whereas hepcidin increased. During the day with iron supplementation the mean TS was significantly higher both in men and in women, and the mean hepcidin was moderately but significantly higher in women (1.0 nmol/L, 95% CI, 0.2-1.8) but not in men (0.0 nmol/L, 95% CI, -0.8 to 0.8). CONCLUSIONS Our data demonstrate that ferritin sets the basal hepcidin concentrations and suggest that innate diurnal rhythm rather than dietary iron mediates the daily hepcidin variations. These findings will be useful for optimizing sampling protocols and will facilitate the interpretation of hepcidin as an iron biomarker.