Hepcidin Status at 2 Months in Infants Fed Breast Milk Compared with Formula.

INTRODUCTION: The basis for the superior absorption of iron from breast milk compared with infant formulas is unclear. The hormone hepcidin downregulates dietary iron absorption. Hepcidin production increases with increased body iron status (reflected in serum ferritin levels). We hypothesized that serum hepcidin levels are suppressed relative to iron status in infants fed breast milk compared with formula. METHODS: Subjects were healthy infants presenting for routine 2-month clinic visit and strictly fed either breast milk or standard infant formula. Urinary hepcidin and ferritin levels (reflective of serum levels) were analyzed and compared across the breast milk- and formula-fed groups. The relationship between urinary hepcidin and ferritin levels within each group was analyzed by linear regression. RESULTS: Twenty-four subjects were enrolled in each group. The median urinary hepcidin level in the group fed breast milk was lower than in formula (130 vs. 359 ng hepcidin/mg creatinine, p < 0.05). However, the median ferritin levels were similar (2.1 vs. 1.9 ng/mL). Within each group, urinary hepcidin correlated with urinary ferritin (r = 0.5, p < 0.05 for each group); however, the slope of the regression line was lower in the group fed breast milk compared with formula (p < 0.005). CONCLUSION: Despite similar urinary ferritin levels, urinary hepcidin levels are lower at 2 months in infants fed breast milk compared with infants fed formula. Hepcidin levels correlate with iron status in each group; however, this relationship is relatively dampened in infants fed breast milk. We speculate that relatively lower infant hepcidin contributes to the superior efficiency of iron absorption from breast milk.

Clinical Immunoassay for Human Hepcidin Predicts Iron Deficiency in First-Time Blood Donors.

BACKGROUND: Serum markers currently used as indicators of iron status have clinical limitations. Hepcidin, a key regulator of iron homeostasis, is reduced in iron deficiency (ID) and increased in iron overload. We describe the first CLIA-validated immunoassay with excellent accuracy and precision to quantify human serum hepcidin. Its diagnostic utility for detecting ID in first-time blood donors was demonstrated. METHODS: A monoclonal competitive ELISA (C-ELISA) was developed for the quantitation of human hepcidin and validated according to CLIA guidelines. Sera from nonanemic first-time blood donors (n = 292) were analyzed for hepcidin, ferritin, transferrin, and serum iron. Logistic regression served to determine the utility of hepcidin as a predictor of ID. RESULTS: The C-ELISA was specific for human hepcidin and had a low limit of quantitation (4.0 ng/mL). The hepcidin concentration measured with the monoclonal C-ELISA was strongly correlated with a previously established, extensively tested polyclonal C-ELISA (Blood 2008;112:4292-7) (r = 0.95, P < 0.001). The area under the receiver operating characteristic curve for hepcidin as a predictor of ID, defined by 3 ferritin concentration thresholds, was >0.9. For predicting ID defined by ferritin <15 ng/mL, hepcidin <10 ng/mL yielded sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin <30 ng/mL yielded sensitivity of 67.6% and specificity of 91.7%. CONCLUSION: The clinical measurement of serum hepcidin concentrations was shown to be a potentially useful tool for diagnosing ID.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Effect of calcitriol on serum hepcidin in individuals with chronic kidney disease: a randomized controlled trial.

BACKGROUND: Anemia is highly prevalent in chronic kidney disease (CKD). Elevated hepcidin concentrations are an important mediator of disordered iron metabolism, a key mechanism underlying anemia of CKD. Vitamin D was recently shown to reduce serum hepcidin concentrations in healthy individuals. We examined whether treatment with calcitriol reduces serum hepcidin in individuals with CKD. METHODS: A total of 40 participants with stage 3 or 4 CKD (eGFR 15-60 ml/min/1.73m2) were randomized to receive either oral calcitriol 0.5 mcg daily or identically-matched placebo for 6 weeks. The primary outcome variable was change in serum hepcidin concentrations. Secondary outcomes variables included the change in iron parameters, calcium, phosphorus, intact parathyroid hormone and hemoglobin concentrations. Study samples were drawn at baseline, 3 days, 1 week, 4 weeks and 6 weeks after randomization. Repeated measures analysis was used to examine differences in outcome variables over time in the two groups. RESULTS: There were no significant differences in the baseline characteristics between the placebo and calcitriol arms. Over 6 weeks of follow-up there were no significant differences in the change in serum hepcidin, iron parameters, or hemoglobin between the two groups. Serum calcium and phosphorus significantly increased and PTH significantly decreased after 6 weeks in calcitriol group whereas these analytes did not change in the placebo group. CONCLUSION: Calcitriol did not reduce serum hepcidin concentrations among individuals with mild to moderate CKD. Future studies are needed to assess if nutritional forms of vitamin D affect hepcidin concentrations in CKD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01988116 . Registered: November 4, 2013.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

A Novel Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitor (GSK1278863) for Anemia in CKD: A 28-Day, Phase 2A Randomized Trial.

BACKGROUND: Anemia associated with chronic kidney disease (CKD) often requires treatment with recombinant human erythropoietin (EPO). Hypoxia-inducible factor-prolyl hydroxylase inhibitors (PHIs) stimulate endogenous EPO synthesis and induce effective erythropoiesis by non-EPO effects. GSK1278863 is an orally administered small-molecule PHI. STUDY DESIGN: Multicenter, single-blind, randomized, placebo-controlled, parallel-group study. SETTING & PARTICIPANTS: Anemic non-dialysis-dependent patients with CKD stages 3-5 (CKD-3/4/5 group; n=70) and anemic hemodialysis patients with CKD stage 5D (CKD-5D group; n=37). INTERVENTIONS: Patients with CKD-3/4/5 received placebo or GSK1278863 (10, 25, 50, or 100mg), and patients with CKD-5D received placebo or GSK1278863 (10 or 25mg) once daily for 28 days. OUTCOMES & MEASUREMENTS: Primary pharmacokinetic and pharmacodynamic (increase and response rates in achieving the target hemoglobin [Hb] concentration, plasma EPO concentrations, reticulocyte count, and others]) and safety and tolerability end points were obtained. RESULTS: Both CKD-3/4/5 and CKD-5D populations showed a dose-dependent increase in EPO concentrations and consequent increases in reticulocytes and Hb levels. Percentages of GSK1278863 participants with an Hb level increase > 1.0g/dL (CKD-3/4/5) and >0.5g/dL (CKD-5D) were 63% to 91% and 71% to 89%, respectively. Per-protocol-defined criteria, high rate of increase in Hb level, or high absolute Hb value was the main cause for withdrawal (CKD-3/4/5, 30%; CKD-5D, 22%). A dose-dependent decrease in hepcidin levels and increase in total and unsaturated iron binding were observed in all GSK1278863-treated patients. LIMITATIONS: Sparse pharmacokinetic sampling may have limited covariate characterization. EPO concentrations at the last pharmacodynamic sample (5-6 hours) postdose may not represent peak concentrations, which occurred 8 to 10 hours postdose in previous studies. Patients were not stratified by diabetes status, potentially confounding vascular endothelial growth factor and glucose analyses. CONCLUSIONS: GSK1278863 induced an effective EPO response and stimulated non-EPO mechanisms for erythropoiesis in anemic non-dialysis-dependent and dialysis-dependent patients with CKD.

Short-Term Effects of Blood Transfusions on Hepcidin in Preterm Infants.

BACKGROUND: Hepcidin, a key regulatory peptide hormone in iron homeostasis, may in future serve as a non-invasive iron status parameter for monitoring iron supplementation in preterm infants. For this, coexisting influencing factors should be taken into account. OBJECTIVES: To evaluate the short-term effects of red blood cell (RBC) transfusions on hepcidin concentrations in serum (HepS) and urine (HepU) of preterm infants. METHODS: This was a prospective, observational study conducted between May 2009 and September 2010 at a single neonatal unit (Tübingen University Hospital, Tübingen, Germany) in very preterm infants, i.e. with a gestational age (GA) of <32 weeks, who received clinically indicated RBC transfusions. The concentration of the mature, 25 amino-acid form of hepcidin was determined in serum und urine by competitive enzyme-linked immunosorbent assay together with cellular indices before and after transfusion. RESULTS: Twenty preterm infants born at a median GA of 26 + 0/7 (interquartile range: 24 + 6/7 to 27 + 3/7) weeks received 27 RBC transfusions at a median corrected age of 31 + 3/7 (29 + 6/7 to 34 + 5/7) weeks. When measured shortly after transfusion (mean time: 10 h), haematocrit values increased from a mean of 26.6% (SD 2.8) to 40.9% (SD 3.2); p < 0.0001. HepS also increased [geometric mean: 44.3 (95% confidence interval 30.8-63.8) ng/ml vs. 58.0 (35.7-94.3) ng/ml; p < 0.05] but HepU remained unaffected. CONCLUSION: The data indicate that HepS concentrations increase shortly after RBC transfusion in preterm infants. Long-term observational studies are needed to understand the dynamics of hepcidin regulation in preterm infants.

Altered erythropoiesis and iron metabolism in carriers of thalassemia.

The thalassemia syndromes (α- and ß-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of α- or ß-globin chain production results in impaired red blood cell synthesis, anemia, and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis, and dysfunctional iron metabolism has not been investigated in both α-thalassemia carriers (ATC) and ß-thalassemia carriers (BTC). Here, we demonstrate that ATC have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, BTC have several hematological parameters significantly different from controls, including increased soluble transferrin receptor and erythropoietin levels. These changes in both groups suggest an altered balance between erythropoiesis and iron metabolism. The index sTfR/log ferritin and (hepcidin/ferritin)/sTfR are, respectively, increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we showed in this study, for the first time in the literature, that thalassemia carriers have altered iron metabolism and erythropoiesis.

Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.

Growth differentiation factor 15 (GDF-15) is a bone marrow-derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE)suggest that hepcidin deficiency mediated by GDF-15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF-15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF-15, and known hepcidin regulators [interleukin-6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO,nor variable GDF-15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF-15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (r(s) = 0.584, P < 0.0001). Our data show that high concentrations of GDF-15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis-driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders.

Hepcidin level predicts hemoglobin concentration in individuals undergoing repeated phlebotomy.

Dietary iron absorption is regulated by hepcidin, an iron regulatory protein produced by the liver. Hepcidin production is regulated by iron stores, erythropoiesis and inflammation, but its physiology when repeated blood loss occurs has not been characterized. Hepcidin was assayed in plasma samples obtained from 114 first-time/reactivated (no blood donations in preceding 2 years) female donors and 34 frequent (≥3 red blood cell donations in preceding 12 months) male donors as they were phlebotomized ≥4 times over 18-24 months. Hepcidin levels were compared to ferritin and hemoglobin levels using multivariable repeated measures regression models. Hepcidin, ferritin and hemoglobin levels declined with increasing frequency of donation in the first-time/reactivated females. Hepcidin and ferritin levels correlated well with each other (Spearman's correlation of 0.74), but on average hepcidin varied more between donations for a given donor relative to ferritin. In a multivariable repeated measures regression model the predicted inter-donation decline in hemoglobin varied as a function of hepcidin and ferritin; hemoglobin was 0.51 g/dL lower for subjects with low (>45.7 ng/mL) or decreasing hepcidin and low ferritin (>26 ng/mL), and was essentially zero for other subjects including those with high (>45.7 ng/mL) or increasing hepcidin and low ferritin (>26 ng/mL) levels (P<0.001). In conclusion, hepcidin levels change rapidly in response to dietary iron needed for erythropoiesis. The dynamic regulation of hepcidin in the presence of a low levels of ferritin suggests that plasma hepcidin concentration may provide clinically useful information about an individual's iron status (and hence capacity to tolerate repeated blood donations) beyond that of ferritin alone. Clinicaltrials.gov identifier: NCT00097006.

Iron homeostasis during cystic fibrosis pulmonary exacerbation.

BACKGROUND: Hypoferremia is a marker of disease severity in cystic fibrosis (CF). The effect of systemic antibiotics on iron homeostasis during CF pulmonary exacerbation (CFPE) is unknown. Our central hypotheses were that, by the completion of treatment, serum iron would increase, serum concentrations of interleukin-6 (IL-6) and hepcidin-25, two mediators of hypoferremia, would decrease, and sputum iron would decrease. METHODS: Blood and sputum samples were collected from 12 subjects with moderate-to-severe CF (median percentage-predicted forced expiratory volume in 1 second (FEV(1) %) = 29%; median weight = 56 kg) within 24 hours of starting and completing a course of systemic antibiotics. RESULTS: After treatment, subjects showed median FEV(1) % and body weight improvements of 4.5% and 2.0 kg, respectively (p < 0.05). Median serum iron rose by 2.4 µmol/L (p < 0.05), but 75% of patients remained hypoferremic. Median serum IL-6 and hepcidin-25 levels fell by 12.1 pg/mL and 37.5 ng/mL, respectively (p < 0.05). Median serum erythropoietin (EPO) and hemoglobin levels were unaffected by treatment. We observed a trend toward lower sputum iron content after treatment. CONCLUSIONS: Hypoferremia is a salient characteristic of CFPE that improves with waning inflammation. Despite antibiotic treatment, many patients remain hypoferremic and anemic because of ineffective erythropoiesis.

Testosterone suppresses hepcidin in men: a potential mechanism for testosterone-induced erythrocytosis.

CONTEXT: The mechanisms by which testosterone increases hemoglobin and hematocrit are unknown. OBJECTIVE: The aim was to test the hypothesis that testosterone-induced increase in hematocrit is associated with suppression of the iron regulatory peptide hepcidin. PARTICIPANTS: Healthy younger men (ages 19-35 yr; n = 53) and older men (ages 59-75 yr; n = 56) were studied. METHODS: Weekly doses of testosterone enanthate (25, 50, 125, 300, and 600 mg) were administered over 20 wk, whereas endogenous testosterone was suppressed by monthly GnRH agonist administration. Blood and serum parameters from each individual were measured at wk 0, 1, 2, 4, 8, and 20. Longitudinal analyses were performed to examine the relationship between hepcidin, hemoglobin, hematocrit, and testosterone while controlling for potential confounders. RESULTS: High levels of testosterone markedly suppressed serum hepcidin within 1 wk. Hepcidin suppression in response to testosterone administration was dose-dependent in older men and more pronounced than in young men, and this corresponded to a greater rise in hemoglobin in older men. Serum hepcidin levels at 4 and 8 wk were predictive of change in hematocrit from baseline to peak levels. CONCLUSION: Testosterone administration is associated with suppression of serum hepcidin. Greater increases in hematocrit in older men during testosterone therapy are related to greater suppression of hepcidin.

Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization.

The recently discovered iron regulatory peptide hormone hepcidin holds promise as a novel biomarker in iron metabolism disorders. To date, various mass spectrometry and immunochemical methods have been developed for its quantification in plasma and urine. Differences in methodology and analytical performance hinder the comparability of data. As a first step towards method harmonization, several hepcidin assays were compared. Worldwide eight laboratories participated in a urinary and plasma round robin in which hepcidin was analyzed. For both urine and plasma: (i) the absolute hepcidin concentrations differed widely between methods, (ii) the between-sample variation and the analytical variation of the methods are similar. Importantly, the analytical variation as percentage of the total variance is low for all methods, indicating their suitability to distinguish hepcidin levels of different samples. Spearman correlations between methods were generally high. The round robin results inform the scientific and medical community on the status and agreement of the current hepcidin methods. Ongoing initiatives should facilitate standardization by exchanging calibrators and representative samples.

Hepcidin--a potential novel biomarker for iron status in chronic kidney disease.

BACKGROUND AND OBJECTIVES: Hepcidin is a key regulator of iron homeostasis, but its study in the setting of chronic kidney disease (CKD) has been hampered by the lack of validated serum assays. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study reports the first measurements of bioactive serum hepcidin using a novel competitive ELISA in 48 pediatric (PCKD2-4) and 32 adult (ACKD2-4) patients with stages 2 to 4 CKD along with 26 pediatric patients with stage 5 CKD (PCKD5D) on peritoneal dialysis. RESULTS: When compared with their respective controls (pediatric median = 25.3 ng/ml, adult = 72.9 ng/ml), hepcidin was significantly increased in PCKD2-4 (127.3 ng/ml), ACKD2-4 (269.9 ng/ml), and PCKD5D (652.4 ng/ml). Multivariate regression analysis was used to assess the relationship between hepcidin and indicators of anemia, iron status, inflammation, and renal function. In PCKD2-4 (R(2) = 0.57), only ferritin correlated with hepcidin. In ACKD2-4 (R(2) = 0.78), ferritin and soluble transferrin receptor were associated with hepcidin, whereas GFR was inversely correlated. In PCKD5D (R(2) = 0.52), percent iron saturation and ferritin were predictors of hepcidin. In a multivariate analysis that incorporated all three groups (R(2) = 0.6), hepcidin was predicted by ferritin, C-reactive protein, and whether the patient had stage 5D versus stages 2 to 4 CKD. CONCLUSIONS: These findings suggest that increased hepcidin across the spectrum of CKD may contribute to abnormal iron regulation and erythropoiesis and may be a novel biomarker of iron status and erythropoietin resistance.

Iron absorption in dysmetabolic iron overload syndrome is decreased and correlates with increased plasma hepcidin.

BACKGROUND/AIMS: The dysmetabolic iron overload syndrome (DIOS) is a common disorder but its origin remains unclear. METHODS: A case-control study was conducted to compare intestinal absorption of iron in 16 men with DIOS (age 53 +/- 11 years, serum ferritin 750 +/- 372 microg/l, hepatic iron 78 +/- 25 micromol/g) and in 32 age-matched controls with normal body iron stores (16 overweight subjects and 16 lean subjects). Intestinal absorption was calculated as the area under the curve (AUC) of 58Fe administered orally and correlated with plasma hepcidin and with insulin resistance parameters including HOMA. RESULTS: Intestinal iron absorption was lower in DIOS (AUC = 22.4 +/- 15.9 microg/l/h) compared to both overweight controls (AUC = 40.5 +/- 29.4 microg/l/h, p=0.04) and to lean controls (AUC = 102.5 +/- 113.5 microg/l/h, p < 0.01). There was an inverse correlation between intestinal iron absorption and plasma hepcidin (r = -0.61, p < 0.001), HOMA (r = -0.35, p = 0.01) and C reactive protein (r = -0.52, p < 0.001). CONCLUSIONS: In overweight subjects with normal iron stores, iron absorption is decreased through hepcidin upregulation. In patients with DIOS, this decrease is more pronounced due to an additional effect of iron excess on circulating hepcidin levels.

Daily regulation of serum and urinary hepcidin is not influenced by submaximal cycling exercise in humans with normal iron metabolism.

Hepcidin and hemojuvelin (HJV) are two critical regulators of iron metabolism as indicated by the development of major iron overload associated to mutations in hepcidin and HJV genes. Hepcidin and HJV are highly expressed in liver and muscles, respectively. Intensive muscular exercise has been reported to modify serum iron parameters and to increase hepcidinuria. The present study aimed at evaluating the potential impact of low intensity muscle exercise on iron metabolism and on hepcidin, its key regulator. Fourteen normal volunteers underwent submaximal cycling-based exercise in a crossover design and various iron parameters, including serum and urinary hepcidin, were serially studied. The results demonstrated that submaximal ergocycle endurance exercise did not modulate hepcidin. This study also indicated that hepcidinuria did not show any daily variation whereas serum hepcidin did. The findings, by demonstrating that hepcidin concentrations are not influenced by submaximal cycling exercise, may have implications for hepcidin sampling in medical practice.

Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young women.

BACKGROUND: Hepcidin is a key regulator of iron homeostasis, but to date no studies have examined the effect of hepcidin on iron absorption in humans. OBJECTIVE: Our objective was to assess relations between both serum hepcidin and serum prohepcidin with nonheme-iron absorption in the presence and absence of food with the use of dual stable-iron-isotope techniques. DESIGN: The study group included 18 healthy nonpregnant women. Women received in random order a supplemental iron source (7.6 mg FeSO4 providing 0.9 mg 58Fe as FeSO4) and 6.8 mg 57Fe ferrous sulfate tracer administered with a nonheme food source [orange-fleshed sweet potato (OFSP): 1.4 mg native Fe]. Iron absorption was determined by analyzing blood samples taken 14 d after dosing with the use of magnetic sector thermal ionization mass spectrometry. Serum hepcidin was assessed by a new competitive serum enzyme-linked immunosorbent assay (ELISA) specific for the refolded, mature 25-amino acid form, and serum prohepcidin was assessed by an ELISA specific for amino acids 28-47 of the hepcidin prohormone. RESULTS: In these women, iron absorption averaged 14.71 +/- 10.7% from the supplemental iron compared with 3.63 +/- 6.5% from the OFSP. Absorption of nonheme iron assessed in the presence (P = 0.038) and absence (P = 0.0296) of food was significantly associated with serum hepcidin but was not significantly related to serum prohepcidin. CONCLUSION: Serum hepcidin, but not prohepcidin, was inversely associated with iron absorption from supplemental and food-based nonheme-iron sources in iron-replete healthy women.

Immunoassay for human serum hepcidin.

We developed and validated the first serum enzyme-linked immunosorbent assay for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. In healthy volunteers, the 5% to 95% range of hepcidin concentrations was 29 to 254 ng/mL in men (n = 65) and 17 to 286 ng/mL in women (n = 49), with median concentrations 112 versus 65 (P < .001). The lower limit of detection was 5 ng/mL. Serum hepcidin concentrations in 24 healthy subjects correlated well with their urinary hepcidin (r = 0.82). Serum hepcidin appropriately correlated with serum ferritin (r = 0.63), reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared with 8 am, and a transient rise of serum hepcidin in response to iron ingestion. Expected alterations in hepcidin levels were observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin < 10 ng/mL), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (C-reactive protein > 10 mg/dL), multiple myeloma, or chronic kidney disease. The new serum hepcidin enzyme-linked immunosorbent assay yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic, and genetic influences, and is informative about the etiology of iron disorders.