Central Regulator Of Iron Metabolism Research Articles

Importance of Hepcidin in the Etiopathogenesis of Anemia in Inflammatory Bowel Disease.

Anemia is the most common extraintestinal systemic complication of inflammatory bowel disease. Iron deficiency anemia and anemia of chronic disease are among the most frequent types. Intestinal iron absorption is controlled by the activity of ferroportin. Cells with high expression of ferroportin include enterocytes, and also macrophages and hepatocytes. Iron homeostasis is controlled by the hepcidin-ferroportin axis. Hepcidin is a central regulator of iron metabolism and can also serve as a marker of systemic inflammation. During systemic inflammatory response, the synthesis of hepcidin increases, and hepcidin binds to ferroportin and inhibits its activity. Thus, iron is not absorbed from the bowel into the circulation and also remains sequestered in macrophages. Conversely, hepcidin synthesis is suppressed during conditions requiring increased iron intake for enhanced erythropoiesis, such as iron deficiency anemia or hypoxia. Here, ferroportin is not blocked, and iron is actively absorbed into the bloodstream and also released from the stores. Production of hepcidin is influenced by the status of total body iron stores, systemic inflammatory activity and erythropoietic activity. Oral iron therapy is limited in inflammatory bowel diseases due to ongoing gastrointestinal inflammation. It is less effective and may worsen the underlying disease. Therefore, the choice between oral and parenteral iron therapy must be made with caution. Oral iron would be ineffective at high hepcidin levels due to concurrent ferroportin blockage. Contrarily, low levels of hepcidin indicate that oral iron therapy should be successful. An understanding of hepcidin can help in understanding the body's reaction to iron depletion during the inflammatory process.

636. The Hepcidin-25 and Iron Kinetics During the Acute Phase of Systemic Infection

BackgroundHepcidin-25, a central regulator of iron metabolism, can decrease serum iron levels by inhibiting the iron transporter ferroportin. Production of hepcidin-25 in hepatocytes is tightly regulated by various stimulations and is promoted by inflammation via the IL-6 pathway. The role of hepcidin-25 in acute infections has not been fully understood; therefore, we investigated the hepcidin and iron kinetics during the acute phase of systemic infection.MethodsWe collected clinical samples of bloodstream infections at various stages and measured plasma hepcidin-25 levels using surface enhanced laser desorption/ionization time-of-flight mass spectrometry. In addition, plasma levels of IL-6, C-reactive protein, procalcitonin, presepsin, lipocalin-2 were measured.ResultsIn this study, 50 patients (median age: 72 years; 52% males) were included. In the acute phase of infection (first 3 days after onset of symptom), plasma hepcidin-25 levels were rapidly elevated, accompanied with a reduction in serum iron concentration. As the inflammation subsequently resolved and the patients’ general condition improved (≥10 days after symptom onset), serum hepcidin-25 levels were decreased and serum iron levels were restored. Therefore, hepcidin-25 and iron levels dynamically vary during the acute phase of infection, and the enhanced production of hepcidin-25 due to severe inflammation can precipitate a rapid decrease of serum iron levels. This series of reactions may be regarded as a host defense involving the inhibition of the nutrient acquirement of bacteria. In this setting, the iron requirement of bacteria is expected to be increased and the iron uptake of bacteria via iron transporter systems may be activated.ConclusionDuring the acute phase of infectious disease with severe inflammation, iron levels were immediately decreased due to enhanced production of hepcidin-25. Understanding of host iron status may be essential for effective use of siderophore cephalosporin, with a unique mechanism of action involving the use of bacterial iron uptake systems.Disclosures All authors: No reported disclosures.

Hepsidin: physiological role how central regulator of iron metabolism

Hepsidin: physiological role how central regulator of iron metabolism

Liver iron is a major regulator of hepcidin gene expression via BMP/SMAD pathway in a rat model of chronic renal failure under treatment with high rHuEPO doses.

Hepcidin is the major central regulator of iron metabolism, controlling iron absorption and mobilization. Considering its interaction with several factors that are altered in chronic kidney disease (CKD), particularly in hyporesponsive CKD patients under therapy with high recombinant human erythropoietin (rHuEPO) doses, it was aimed to study the impact of increasing rHuEPO doses on the regulation of iron-hepcidin metabolism. The blood, cellular, and tissue studies, using the remnant kidney rat model of CKD induced by 5/6 nephrectomy, under rHuEPO (100, 200, 400, and 600 IU/Kg body weight [BW]/week) treatment during 3 weeks were performed. It was found that the rHuEPO stimulus triggered a first wave to achieve correction of anemia, by inhibiting hepcidin synthesis, favoring erythropoiesis and iron absorption; this continuous stimulus enhanced iron absorption leading to iron overload, as showed by the hepatic iron deposits found in rats treated with higher rHuEPO dose that seems to trigger the upregulation of hepcidin synthesis through the activation of the BMP6/SMAD pathway. The data suggested that liver iron overload was an important stimuli for hepcidin synthesis, stronger than the inhibitory effect of high rHuEPO doses; moreover, the findings raised the hypothesis that when high inflammation (triggering hepcidin expression) was associated with increased iron stores in hemodialysis patients, hepcidin expression was also upregulated via BMP6, enhancing hepcidin synthesis, leading, therefore, to worsening of anemia and, eventually, to a hyporesponse/resistance to rHuEPO therapy. © 2016 BioFactors, 42(3):296-306, 2016.

Transferrin Receptor 2 and HFE Regulate Hepcidin Levels Independently of Bmp6 and Hemojuvelin

Hereditary hemochromatosis (HH) is a genetically heterogeneous disorder characterized by elevated iron absorption from the diet, with consequent iron overload and tissue injury. Adult-onset forms of HH are caused by mutations in the HFE gene and in the gene for transferrin receptor 2 (TFR2). Human patients and mouse models of TFR2 and HFE-related HH show inappropriately low expression of hepcidin, the central regulator of iron metabolism. However, although these genes have been discovered far more than a decade ago, the mechanisms by which HFE and TFR2 influence hepcidin expression remain unclear.The bone morphogenetic protein BMP6 plays a key role in the regulation of hepcidin expression. BMP6 binds to type I (ALK3) and type II serine threonine kinase receptors, and to the coreceptor hemojuvelin (HJV), which phosphorylates intracellular SMAD proteins. Phosphorylated SMADs then bind to SMAD4 and translocate to the nucleus to induce the transcription of hepcidin. Inactivation of Bmp6 or Hjv in mice leads to considerably reduced hepcidin production and severe hepatic iron overload. However, there are major differences in hepcidin expression and extrahepatic tissue iron loading between Bmp6 or Hjv KO males and females, due to the suppressive effect of testosterone on hepcidin in males. In contrast to males, Bmp6-/- and Hjv-/-females still produce some hepcidin and do not massively accumulate iron in the pancreas, heart, or kidneys.The goal of this study was to investigate the role of Hfe and Tfr2 in the residual hepcidin production observed in the absence of Bmp6 in females. We used Bmp6-/-, Tfr2-/-, and B2m-/- mice to generate wild-type, single KO (Bmp6-/-, Tfr2-/-, or B2m-/-) and double KO (Tfr2-/- and Bmp6-/-, or B2m-/- and Bmp6-/-) mice, and we assessed Smad5 phosphorylation, hepcidin expression, and the sites of iron accumulation in the different groups of mice. Notably, B2m-/- mice develop spontaneously hepatic iron overload with a distribution similar to that seen in the liver of Hfe-/-mice and the lack of CD8+ lymphocytes and the absence of classical class I molecules in these mice are not responsible for their iron phenotype.Interestingly, the lack of functional Hfe or the lack of Tfr2 in Bmp6-/- females leads to a very similar phenotype that is much more severe than the single impairment of Bmp6, with massive iron loading in extrahepatic tissues, most notably the exocrine pancreas, the heart, and the kidney. Hepcidin mRNA and pSmad levels in the two categories of double KO females were much more strongly downregulated than in single Bmp6-/- females and, in contrast to Bmp6-/-females, no protein was detectable by ELISA in the double KO mice.Our findings clearly demonstrate that Hfe and Tfr2 regulate hepcidin production independently of Bmp6. The symmetrical phenotype of double Bmp6 and Tfr2, or double Bmp6 and Hfe KO mice suggests that Hfe and Tfr2 could participate in the same signaling complex affecting pSmad levels, even if they do not physically interact. Two complexes, one made of ALK3 and HJV, and the other made of ALK2, ALK3, HFE, and TFR2, very likely affect pSMAD levels independently of each other. Signaling through the second complex occurs in the absence of BMP6 and is most probably initiated by another BMP, for instance BMP2 or BMP4 that are also expressed in the liver and, although not regulated by iron, are capable of stimulating hepcidin expression in hepatocytes. Regulation of hepcidin expression by these two complexes would explain why, whereas Alk3, double Bmp6/Hfe, and double Bmp6/Tfr2 deficient females present with iron overload in extrahepatic tissues as do hepcidin KO mice, Bmp6 and Hjv single KO females present with early-onset iron overload only in the liver, similarly to patients with juvenile HH, and mice KO for Alk2, Hfe, or Tfr2 have the least severe phenotype, comparable with that of patients with adult-onset HH. Notably, TFR2 was also shown to localize in lipid rafts where it promotes MAPK activation. Thus, in addition to its role in the complex affecting pSMAD levels, TFR2 could also regulate hepcidin through a parallel pathway involving ERK1/2 signaling. This would explain the more severe phenotype of mice with combined deletion of Hfe and Tfr2 and the fact that, whereas Tfr2-/- mice do not respond to acute iron loading, Hfe-/-mice still have a significant, although blunted hepcidin response.This work was funded in part by FRM (DEQ2000326528) and ANR (ANR-13-BSV3-0015-01). DisclosuresNo relevant conflicts of interest to declare.

CHOP‐mediated hepcidin suppression modulates hepatic iron load

The liver is the central regulator of iron metabolism and accordingly, chronic liver diseases often lead to systemic iron overload due to diminished expression of the iron-regulatory hormone hepcidin. To study the largely unknown regulation of iron metabolism in the context of hepatic disease, we used two established models of chronic liver injury, ie repeated carbon tetrachloride (CCl(4)) or thioacetamide (TAA) injections. To determine the impact of CCAAT/enhancer-binding protein (C/EBP)-homologous protein (CHOP) on hepcidin production, the effect of a single TAA injection was determined in wild-type and CHOP knockout mice. Furthermore, CHOP and hepcidin expression was assessed in control subjects and patients with alcoholic liver disease. Both chronic injury models developed a distinct iron overload in macrophages. TAA-, but not CCl(4) - injected mice displayed additional iron accumulation in hepatocytes, resulting in a significant hepatic and systemic iron overload which was due to suppressed hepcidin levels. C/EBPα signalling, a known hepcidin inducer, was markedly inhibited in TAA mice, due to lower C/EBPα levels and overexpression of CHOP, a C/EBPα inhibitor. A single TAA injection resulted in a long-lasting (> 6 days) suppression of hepcidin levels and CHOP knockouts (compared to wild-types) displayed significantly attenuated hepcidin down-regulation in response to acute TAA administration. CHOP mRNA levels increased 5-fold in alcoholic liver disease patients versus controls (p < 0.005) and negatively correlated with hepcidin expression. Our results establish CHOP as an important regulator of hepatic hepcidin expression in chronic liver disease. The differences in iron metabolism between the two widely used fibrosis models likely reflect the differential regulation of hepcidin expression in human liver disease.

Anemia of Chronic Disease

Anemia of chronic disease (ACD) or inflammation may be secondary to infections, autoimmune disorders, chronic renal failure, or malignancies. It is characterized by an immune activation with an increase in inflammatory cytokines and resultant increase in hepcidin levels. In addition, inappropriate erythropoietin levels or hyporesponsiveness to erythropoietin and reduced red blood cell survival contribute to the anemia. Hepcidin being the central regulator of iron metabolism plays a key role in the pathophysiology of ACD. Hepcidin binds to the iron export protein, ferroportin, present on macrophages, hepatocytes, and enterocytes, causing degradation of the latter. This leads to iron trapping within the macrophages and hepatocytes, resulting in functional iron deficiency. Production of hepcidin is in turn regulated by iron stores, inflammation, and erythropoiesis via the BMP-SMAD and JAK-STAT signaling pathways. Treatment of anemia should primarily be directed at the underlying disease, and conventional therapy such as red blood cell transfusions, iron, erythropoietin, and novel agents targeting the hepcidin-ferroportin axis and signaling pathways (BMP-SMAD, JAK-STAT) involved in hepcidin production also may be considered.

Tubular reabsorption and local production of urine hepcidin-25

BackgroundHepcidin is a central regulator of iron metabolism. Serum hepcidin levels are increased in patients with renal insufficiency, which may contribute to anemia. Urine hepcidin was found to be increased in some patients after cardiac surgery, and these patients were less likely to develop acute kidney injury. It has been suggested that urine hepcidin may protect by attenuating heme-mediated injury, but processes involved in urine hepcidin excretion are unknown.MethodsTo assess the role of tubular reabsorption we compared fractional excretion (FE) of hepcidin-25 with FE of β2-microglobulin (β2m) in 30 patients with various degrees of tubular impairment due to chronic renal disease. To prove that hepcidin is reabsorbed by the tubules in a megalin-dependent manner, we measured urine hepcidin-1 in wild-type and kidney specific megalin-deficient mice. Lastly, we evaluated FE of hepcidin-25 and β2m in 19 patients who underwent cardiopulmonary bypass surgery. Hepcidin was measured by a mass spectrometry assay (MS), whereas β2m was measured by ELISA.ResultsIn patients with chronic renal disease, FE of hepcidin-25 was strongly correlated with FE of β2m (r = 0.93, P <0.01). In megalin-deficient mice, urine hepcidin-1 was 7-fold increased compared to wild-type mice (p < 0.01) indicating that proximal tubular reabsorption occurs in a megalin- dependent manner. Following cardiac surgery, FE of hepcidin-25 increased despite a decline in FE of β2m, potentially indicating local production at 12–24 hours.ConclusionsHepcidin-25 is reabsorbed by the renal tubules and increased urine hepcidin-25 levels may reflect a reduction in tubular uptake. Uncoupling of FE of hepcidin-25 and β2m in cardiac surgery patients suggests local production.

Hepcidin: another culprit for complications in patients with chronic kidney disease?

Hepcidin has been established as a central regulator of iron metabolism. In most patients with chronic kidney disease (CKD), serum hepcidin levels are relatively high, favoring iron sequestration in several cell types and organs and thereby leading to iron-related complications. In the absence of overt inflammation, serum hepcidin has been found to be most closely associated with serum ferritin in healthy subjects and in CKD patients. Intestinal iron absorption is tightly regulated by both iron stores and hepcidin. The expression of the mammalian iron exporter, ferroportin (FPN), limits the growth of intracellular bacteria by depleting cytosolic iron. An upregulation of hepcidin could diminish FPN and favor bacterial growth. Of note, in patients with hyperferritinemia impaired hepcidin expression caused by a mutation in the hemochromatosis gene associates with an attenuation of atherosclerosis. Thus, hepcidin might accelerate atherosclerosis by preventing iron exit from macrophages or other cells in the arterial wall. High hepcidin levels have also been found to be linked to good erythropoiesis-stimulating agents (ESAs) response, in conjunction with the strong hepcidin-ferritin correlation. Finally, hepcidin may also play a significant role by itself in the pathogenesis of CKD complications associated with disturbed iron metabolism, i.e. unrelated to ESA hyporesponsiveness, such as bacterial infections and atherosclerosis.

Hepcidin is localised in gastric parietal cells, regulates acid secretion and is induced by Helicobacter pylori infection

Backgrounds and aimsHepcidin is an antimicrobial peptide and the central regulator of iron metabolism. Given that hepcidin was shown to be expressed in a variety of extrahepatic tissues and that...

Impaired expression of duodenal iron transporters in Dahl salt-sensitive heart failure rats

Anemia is common in patients with heart failure and several factors have been thought to cause anemia in heart failure. Despite vigorous studies, the mechanism underlying the pathophysiology of anemia in heart failure is unknown. We investigated the iron regulating system in Dahl salt-sensitive heart failure rats to elucidate the mechanism of anemia in heart failure. Dahl salt-sensitive rats were provided either a normal or high-salt diet to initiate heart failure progression. A further subset of Dahl salt-sensitive rats underwent an iron-deficient diet to induce iron deficiency anemia (IDA). Dahl salt-sensitive rats, which develop diastolic heart failure, gradually showed hypertension and anemia after 8 weeks of high-salt diet. Although serum iron levels were decreased, erythropoietin levels were increased in the IDA and heart failure groups. Hepatic expression of hepcidin, a central regulator of iron metabolism, was downregulated in both IDA and heart failure groups. Duodenal cytochrome b (Dcyt-b), divalent metal transporter 1 (DMT-1), and ferroportin are the crucial regulators of intestinal iron transport and absorption. Duodenal expression levels of these molecules were markedly upregulated in the IDA group, but not in the heart failure group. Moreover, intestinal expression of hypoxia-inducible factor-2α, a critical regulator of the transcription of Dcyt-b and DMT-1, was upregulated in the IDA group, but not in the heart failure group. Duodenal iron transporters expression was impaired in Dahl heart failure rats. Our data suggest that impaired duodenal iron absorption may occur in Dahl heart failure rats. Understanding the mechanism of abnormal iron regulating system may lead to new therapeutic strategies in anemia with heart failure.

Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice

AbstractAs a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Urinary hepcidin: an inverse biomarker of acute kidney injury after cardiopulmonary bypass?

In this review, we discuss the potential role of urinary hepcidin, a 2.8-kDa hormonal regulator of iron metabolism, as a biomarker of acute kidney injury (AKI) after cardiopulmonary bypass. Hepcidin is one of the novel biomarkers of AKI that have been identified using hypothesis-free, proteomic analysis of urine or plasma in patients who develop AKI. Collectively, these markers promise a new era for the early diagnosis and treatment of AKI in the ICU and an understanding of their biological role may also provide mechanistic insights into the pathogenesis of AKI. Although data confirming the association between urinary hepcidin and AKI are as yet limited, we believe hepcidin is of particular interest because hepcidin may be a biomarker specific to cardiopulmonary bypass-associated AKI; as a central regulator of iron metabolism, hepcidin could play a biological role in the pathogenesis of AKI after cardiopulmonary bypass; and hepcidin displays an intriguing negative association with AKI, in that a smaller increase in hepcidin from baseline after cardiopulmonary bypass appears to predict greater chance of developing AKI. Smaller increases in urinary hepcidin, a central regulator of iron metabolism, may be associated with greater risk of AKI after cardiopulmonary bypass. Further research is required to establish the significance and nature of this association.

Role of iron metabolism-regulator hepcidin in perinatal iron homeostasis

Hepcidin is a recently recognized defensin-like peptide, which is considered to be the central regulator of iron metabolism. Hepcidin decreases the expression of iron transporting molecules. Hepcidin reduces gastrointestinal iron absorption, iron release from the macrophages, and hence it decreases serum iron levels. Clarification of hepcidin role in iron homeostasis could provide an explanation to anemia of inflammation and chronic diseases. At start of our work there was no commercially available method for measuring urine hepcidin levels. The aim of our study was to develop an easily achievable, reliable quantification method for the determination of urine hepcidin levels in human, in addition to examine a possible association of hepcidin with neonatal iron homeostasis. According to the sequence of native, human hepcidin we have synthesized peptide derivatives from which 1-7 peptide derivatives might be suitable representatives of the 25-amino-acid form of hepcidin in immune adsorption method. We presented a novel laser-desorption mass spectrometry based semi-quantitative, reproducible method for measuring hepcidin concentration in human urine first using the synthesized peptide derivative acetyl-1-25 peptide as hepcidin related internal standard. We described an easy and quickly achievable solid-phase extraction method which is suitable for purification of urine and concentration of hepcidin. In our study we have first measured serum prohepcidin and urine hepcidin in healthy human newborns. Serum prohepcidin levels showed no significant changes, however, urine hepcidin levels increased significantly during the first postnatal days. Serum prohepcidin and urine hepcidin levels showed no significant association in healthy human newborns. Associations have been demonstrated between cord blood prohepcidin values and mean corpuscular hemoglobin concentration as well as between urine hepcidin levels and serum iron and total iron binding capacity values. We have demonstrated that neonates with detectable non-protein-bound iron levels in cord blood were presented with lower prohepcidin concentrations. In summary, our results suggest a possible link between hepcidin and early iron adaptation of newborn's, however, further investigations should be done to elucidate this issue.

Hepcidin as a Predictor of Response to Epoetin Therapy in Anemic Cancer Patients

Hepcidin is thought to be the central regulator of iron metabolism. Iron deficiency is associated with low hepcidin concentrations, and anemia in patients with cancer is associated with high concentrations of hepcidin. Our main objective was to assess the potential role of hepcidin for predicting response to epoetin therapy in anemic cancer patients. We also aimed to identify a cutoff value for hepcidin as a potential predictive marker for response to epoetin therapy. Using data from 525 anemic cancer patients enrolled in 5 studies, we assessed serum hepcidin concentrations in 408 of these patients at baseline and analyzed pooled data from the 408 patients. The analysis population was separated into 2 categories using a threshold hepcidin concentration of 13 nmol/L: low hepcidin (< 13 nmol/L) and high hepcidin (> or = 13 nmol/L). A significantly higher percentage of responders (defined as hemoglobin increase > or = 10 g/L or > or = 20 g/L from baseline) was observed in the low hepcidin group compared with the high hepcidin group (P = 0.04 for > or = 10 g/L increase and P = 0.009 for > or = 20 g/L from baseline). There was also a statistically significant difference between the 2 groups for hematopoietic response (hemoglobin rise at least once > or = 20 g/L from baseline or at least once > or = 120 g/L) to epoetin therapy (P = 0.0004). The results of this analysis suggest a potential role of hepcidin serum concentrations in predicting the response to epoetin therapy.

Serum prohepcidin concentrations in rheumatoid arthritis

Hepcidin, a recently identified peptide, acts as a central regulator of iron metabolism. It is regarded as a factor regulating the uptake of dietary iron and its mobilisation from macrophages and hepatic stores. It is considered as a mediator of anaemia of inflammation. The aim of this study was to assess whether serum prohepcidin concentration is able to distinguish iron deficiency from anaemia of inflammation in patients with rheumatoid arthritis (RA). Blood samples were obtained from 20 healthy blood donors, 30 RA patients who presented with anaemia and 30 patients who had pure iron deficiency anaemia (IDA). The samples were analysed for full blood count, iron, ferritin, transferrin, soluble transferrin receptor and prohepcidin. The mean prohepcidin level in the control subjects was 256 microg/L. The prohepcidin level was significantly lower in IDA patients (100 microg/L; p < 0.0001) but not significantly different from that of control in RA patients (250 microg/L; p > 0.05). Higher serum soluble transferrin receptor (sTfR) levels were observed in IDA (p < 0.0001) but not in RA compared with that of control (p > 0.05). RA patients were divided into iron depleted and iron repleted subgroups based on the ferritin level. Prohepcidin in the iron depleted group was significantly lower than the iron repleted group and the control (p < 0.0001) and higher levels were observed in the iron repleted group (p < 0.01). sTfR levels in the iron depleted group were significantly higher than the control and the iron repleted patients (p < 0.001). In the iron repleted group, sTfR level was not statistically different from that of control (p > 0.05). Serum prohepcidin is clearly reduced in uncomplicated iron deficiency anaemia. The reduced prohepcidin levels in the iron depleted RA patients suggests that there may be conflicting signals regulating hepcidin production in RA patients. In RA patients who have reduced hepcidin in the iron depleted group (ferritin <60 microg/L) where sTfR levels are increased suggests that these patients are iron deficient. Further studies with a larger cohort of patients are required to substantiate this point.

Quantification of Hepcidin-25 in Human Serum by Isotope Dilution Micro-HPLC–Tandem Mass Spectrometry

Hepcidin-25, a liver-produced peptide hormone, was initially isolated from human urine and blood ultrafiltrate. Hepcidin-25 is thought to be the central regulator of iron metabolism (1). Iron deficiency is associated with low hepcidin-25 concentrations and anemia of chronic disease with high concentrations, but the true diagnostic value of hepcidin-25 is still under investigation. A recently published review(2) stated that only semiquantitative methods for comparative studies based on mass spectrometry (MS) have been used for the determination of hepcidin in serum and urine. Isotope-dilution MS is generally accepted as yielding high analytical specificity and accuracy. We report an isotope-dilution–micro-HPLC–tandem MS (MS/MS) method that allows the quantification of hepcidin-25 present at less than nanomole-per-liter concentrations. During sample preparation, hepcidin-25 undergoes strong nonspecific binding to surfaces. A stable isotope-labeled internal standard helps to compensate for any matrix effects. Micro-HPLC-MS/MS with monolithic capillary columns ensures highest resolution and limits of quantification (3). Native hepcidin-25 ( M r 2789) was purchased from Bachem AG. The internal standard, hepcidin-25 [DTHFPI(13C6 …

Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry

AbstractThe hepatic peptide hormone hepcidin is considered the central regulator of iron metabolism. Characterizing the circulating levels of this peptide is critical to understanding its role in the development of clinically relevant syndromes, such as anemia of inflammation/chronic disease, and may provide insight into potential clinical interventions. While quantitative methods have been published for the determination of urinary hepcidin and serum prohepcidin, no definitive methods have been published for the determination of hepcidin in serum. In this report, we describe a quantitative method for the determination of both human and mouse hepcidin in serum and plasma. The method employs protein precipitation and solid-phase extraction followed by liquid chromatographic separation and tandem mass spectrometry detection. The method has a quantitative range of 0.25 ng/mL to 500 ng/mL serum for mouse hepcidin and 1 ng/mL to 500 ng/mL serum for human hepcidin. The method uses small sample volumes (50 μL for mice and 100 μL for humans) and 96-well formats for rapid sample processing. The method was used to establish baseline serum and plasma concentrations of hepcidin in normal C57Bl/6 mice and healthy human volunteers.

Hepcidin – central regulator of iron metabolism

The knowledge about mammalian iron metabolism has advanced dramatically over the past decades. Studies of genetics, biochemistry and molecular biology allowed us the identification and characterization of many of the molecules involved in regulation of iron homeostasis. Important progresses were made after the discovery in 2000 of a small peptide--hepcidin--that has been proved to play a central role in orchestration on iron metabolism also providing a link between iron metabolism and inflammation and innate immunity. Hepcidin directly interacts with ferroportin (FPN), the only known mammalian iron exporter, which is expressed by enterocytes, macrophages and hepatocytes. The direct hepcidin-FPN interaction allows an adaptative response from the body in situations that alter normal iron homeostasis (hypoxia, anemia, iron deficiency, iron overload, and inflammation).

Novel urine hepcidin assay by mass spectrometry

AbstractThe hepatic peptide hormone hepcidin is the central regulator of iron metabolism and mediator of anemia of inflammation. To date, only one specific immuno-dot assay to measure hepcidin in urine had been documented. Here we report an alternative approach for quantification of hepcidin in urine by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Peptide peaks were detected corresponding to the 3 forms of hepcidin normally found in urine. The identity of the peptide peak equivalent to hepcidin-25 was confirmed using synthetic human hepcidin-25. Validation of our MS data on samples with various hepcidin levels showed a strong correlation with previous immuno-dot assay results (Spearman R = 0.9275, P < .001). Most importantly, this hepcidin assay clearly discriminates between relevant clinical iron disorders. In conclusion, this novel MS urine hepcidin assay is easy to perform and available to a wide audience. This enables the implementation of hepcidin measurements in large clinical studies.