Hepcidin in human iron disorders: diagnostic implications.

Physiol Res

2006;55:667-74.



(182.) Tussing-Humphreys LM, Nemeth E, Fantuzzi G, Freels S,

Holterman AX, Galvani C, et al. Hereditary

hemochromatosis: genetic complexity and new diagnostic approaches. AAPS J 2010;12:646-57.



(23.) Merle U, Fein E, Gehrke SG, Stremmel W, Kulaksiz H. Hepcidin-25 induces the internalization and degradation of ferroportin (35-38), resulting in increased

intracellular iron stores, decreased dietary iron absorption, and

decreased circulating iron concentrations (Fig. Kidney Int 2009; 75:976-81.



(187.) Lapenna D, Pierdomenico SD, Ciofani G, Ucchino S, Neri M,

Giamberardino MA, Cuccurullo F. 4). pylori-induced

inflammation does not influence iron status through increased hepcidin

production in childhood (118).



(34.) Wan L, Bellomo R, Di GD, Ronco C. PLoS ONE 2009;4:e6618.



(157.) Cercamondi CI, Egli IM, Ahouandjinou E, Dossa R, Zeder C,

Salami L, et al. J Clin Invest 2009;119:1159-66.



Honoraria: None declared.



The development and validation of hepcidin assays paved the way for

a large number of human studies that considerably increased our

understanding of the physiology and pathophysiology of iron homeostasis

and moreover demonstrated promising applications for hepcidin in

diagnostic medicine (Table 2).



(48.) Finberg KE, Whittlesey RL, Fleming MD, Andrews NC.

Down-regulation of Bmp/Smad signaling by Tmprss6 is required for

maintenance of systemic iron homeostasis. Blood 2009;113: 6225-36.



In a proof-of-principle study, Sasu et al. Serum hepcidin levels are innately low in

HFE-related haemochromatosis but differ between C282Y-homozygotes with

elevated and normal ferritin levels. Clinical stability, time of sampling in relation to iron and ESA

therapy, and iron and EPO dosage and dialysis regimens differ between

studies and are likely to influence results. Serum prohepcidin concentration: no association with iron

absorption in healthy men; and no relationship with iron status in men

carrying HFE mutations, hereditary haemochromatosis patients undergoing

phlebotomy treatment, or pregnant women. Hepcidin, a new iron regulatory peptide. Nat Genet 2006;38:531-9.



Expert Testimony: None declared.



(221.) Klaus S, Langsetmo I, Spong S, Gervasi B, Nichols B, Fourney

P, et al. Blood Cells Mol Dis 2008;40: 347-52.



Consultant or Advisory Role: D.W. Br J Haematol

2009;145:657-64.



(107.) Kroot JJ, Hendriks JC, Laarakkers CM, Klaver SM, Kemna EH,

Tjalsma H, Swinkels DW. A new mouse liverspecific gene, encoding a protein

homologous to human antimicrobial peptide hepcidin, is over-expressed

during iron overload. This effect might be attributable in part to

the effect of hypoxia on EPO expression and thus erythropoietic

activityand/or possibly via a direct interaction with hepatocyte

receptors (51). J Hepatol

2009;50:1219-25.



Administration of erythropoiesis-stimulating agents (ESA) was found

to decrease (hepatocyte) hepcidin production in murine, human, and in

vitro studies (50-53). Patients with cancer often suffer from anemia,

generally in association with other markers of inflammation. Haematologica 2007;92: 857-8.



(186.) Kraml PJ, Klein RL, Huang Y, Nareika A, Lopes-Virella MF.

Iron loading increases cholesterol accumulation and macrophage scavenger

recep tor I expression in THP-1 mononuclear phagocytes. First, any postexercise free iron uptake by the RE

macrophages may be trapped within the scavenger cell owing to the action

of hepcidin on the ferroportin transporters, thereby affecting the

recycling of iron back into the circulation. Although no specific hepcidin

therapies are available, several compounds are under development as

hepcidin agonists or antagonists [reviewed in (204-206)]. Am J

Trop Med Hyg 2007;77:623-6.



The quantification of hepcidin has been found to be complicated by

its tendency to aggregate (79) and to stick to laboratory plastics,

necessitating implementation of robust laboratory procedures.

Furthermore, progress in developing a conventional immunochemical hepcidin assay has been hampered by difficulties in generating specific

antihepcidin antibodies in hosts such as rabbits. However, circadian variation in hepcidin could

be secondary as well, because it might be driven by the influence of

iron intake during the day. The hepatocellular interaction of IL-6

with its receptor activates the janus kinase/signal transducer and

activator of transcription-3 signaling pathway (59,64-66). Blood

2010;115:3616-24.



(153.) de Mast Q, Nadjm B, Reyburn H, Kemna EH, Amos B, Laarakkers

CM, et al. Biochim Biophys Acta 2009;1790:718-23.



(61.) Silvestri L, Pagani A, Camaschella C. Serum hepcidin concentration in chronic

haemodialysis patients: associations and effects of dialysis, iron and

erythropoietin therapy. Blood Cells Mol Dis 2010;45: 238-45.



(170.) van der Putten K, Jie KE, van den Broek D, Kraaijenhagen RJ,

Laarakkers C, Swinkels DW, et al. Blocking HIF

could also be of useful for increasing hepcidin concentrations in

iron-overload diseases.



(97.) Grebenchtchikov N, Geurts-Moespot AJ, Kroot JJ, den Heijer M,

Tjalsma H, Swinkels DW, Sweep FG. Tjalsma, Radboud University Ni jmegen

Medical Centre (among others offers services through

hepcidinanalysis.com); D.W. However, the use of

different assay technologies to assess hepcidin concentrations in

patients with disorders of iron metabolism makes it difficult to

interpret the results of the many reported studies. Mass spectrometry-based proteomic analysis of urine in acute kidney

injury following cardiopulmonary bypass: a nested case-control study. (89)

MALDI-TOF MS Kroot et al. Hepcidin regulates cellular iron efflux by binding to

ferroportin and inducing its internalization. Swinkels, Radboud University Nijmegen Medical Centre (among others offers services through

hepcidinanalysis.com).



(203.) Peeling P. 2) (12-17). Clin Chem 2010;56:1725-32.



Hepcidin Methods



(160.) Weiss G, Goodnough LT. Regulation of hepcidin and iron-overload

disease. Erythroid activity, transfusion iron overload, and

hepcidin levels in patients with myelodysplastic syndrome. An insight into the relationships between

hepcidin, anemia, infections and inflammatory cytokines in pediatric

refugees: a cross-sectional study. vivax

malaria. The iron

regulatory peptide hepcidin is expressed in the heart and regulated by

hypoxia and inflammation. The relevance of these studies is questionable,

because of the fact that prohepcidin concentrations correlate with

neither urinary nor serum hepcidin concentrations, nor do they correlate

with relevant physiological responses (71, 81-83). Elderly people often suffer from anemia. Adv Hematol 2010;2010:750643.



REGULATION BY INFLAMMATION



Hepcidin in infectious and inflammatory diseases. These

include kidney tubule, heart, retina, monocytes, neutrophils, fat cells,

alveolar cells, pancreatic [beta]-cells, and cardiomyocardal cells

(20-27). Expression and localization of hepcidin in macrophages: a

role in host defense against tuberculosis. Crit

Care Med 2008;36:2388-94.



(57.) Chou ST, Weiss MJ. Plos One 2008;3:e4030



(12.) Kemna EH, Tjalsma H, Podust VN, Swinkels DW. Results are expected during 2011

(unpublished data).



(109.) Galesloot TE, Vermeulen SH, Geurts-Moespot AJ, Klaver SM,

Kroot JJ, van Tienoven D, et al. Targeting the hepcidin-ferroportin axis in the

diagnosis and treatment of anemias. Clin Chem 2007;53: 620-8.



(132.) Girelli D, Trombini P, Busti F, Campostrini N, Sandri M,

Pelucchi S, et al. It is possible that binding to [alpha]2-macroglobulin or

other carrier proteins prevents circulating hepcidin from being

freelyfiltered. Serum hepcidin is significantly associated

with iron absorption from food and supplemental sources in healthy young

women. J

Endocrinol 2005;184:361-70.



(41.) Theurl I, Theurl M, Seifert M, Mair S, Nairz M, Rumpold H, et

al. reported that transgenic hepcidin overexpression in

[beta]-thalassemia intermedia mice decreased iron overload, and even

ameliorated ineffective erythropoiesis (207).



Several reports suggest the potential use of hepcidin

concentrations in determining cardiovascular risk. The hypothesis that

erythropoietin (EPO) acts directly on hepatocyte receptors in cell

culture (51) could not be confirmed in animal models for anemia, which

showed that decreased hepcidin expression depends on erythropoiesis and

is not directly mediated by EPO (53, 54).



(44.) Hentze MW, Muckenthaler MU, Galy B, Camaschella C. STAT3 mediates hepatic hepcidin expression and its

inflammatory stimulation. Am J Physiol Lung

Cell Mol Physiol 2006;291:L417-25.



(197.) Ukarma L, Johannes H, Beyer U, Zaug M, Osterwalder B,

Scherhag A. Effect of body mass index reduction

on serum hepcidin levels and iron status in obese children. The

former defect leads to greater than normal ferroportin activity and thus

is often described as a "gain of function" ferroportin

mutation. Kidney Int 2010;78:769-73.



(63.) Mastrogiannaki M, Matak P, Keith B, Simon MC, Vaulont S,

Peyssonnaux C. J Clin Oncol 2011;29:e42.



Hepcidin has been demonstrated to be important in the pathogenesis

of other infectious agents as well. Hepcidin in obese children as a potential mediator

of the association between obesityand iron deficiency. (91)

SELDI-TOF MS Campostrini et al. Virtually all studies on hepcidin in CKD patients

revealed a strong positive relationship between hepcidin and ferritin

concentrations. Some studies report a weak, but

significant, negative correlation between serum hepcidin concentrations

and iron absorption (112, 146, 147). These conditions

include iron deficiency, hypoxia, anemia, and conditions characterized

by increased erythropoietic activity. This finding raises the

possibility that in this population hepcidin-independent mechanisms can

cause hypoferremia and anemia of inflammation, and/or that even

otherwise "normal" hepcidin concentrations are sufficient to

sustain anemia once it is initiated (162).



(101.) Kiernan UA, Tubbs KA, Gruber K, Nedelkov D, Niederkofler EE,

Williams P, Nelson RW. Possibly the consequent increase in hepcidin by

inflammatory signals attenuates iron toxicity than would otherwise be

observed in sickle cell disease patients (141, 143).



(100.) Butterfield AM, Luan P, Witcher DR, Manetta J, Murphy AT,

Wroblewski VJ, Konrad RJ. Hepcidin

expression is also increased byoxidative or endoplasmic reticulum stress. A number of exercise-generated mechanisms may

contribute to this low iron status of athletes, including hemolysis,

hematuria, sweating, and gastrointestinal bleeding (198). Other

groups have also reported reliable hepcidin assays that can be divided

in 3 main methodologies: (a)MS(85-95);(b) immunochemical assays,

comprising cRIA (96, 97), cELISA (13, 29, 98, 99), and a 2-site ELISA

(100); and (c) a ligand-binding assay (43) (Table 1). Perhaps, in such areas, untargeted

iron supplementation among iron-replete children might result in severe

adverse events by increasing the proliferation of latent pathogens

(158).



(205.) Pietrangelo A. Blood

2008;112:5241-4.



It is speculative, but conceivable, that under certain conditions

hepcidin escapes renal tubular reabsorption. These findings of increased

hepcidin concentrations led Peeling et al. Increased serum hepcidin and alterations in blood iron

parameters associated with asymptomatic P. Cumulative effects of consecutive running

sessions on hemolysis, inflammation and hepcidin activity. Moreover, in a previous study we found a relatively high

(pre)analytical variation for urine hepcidin measurements (107). Expression of the peptide hormone hepcidin increases in

cardiomyocytes under myocarditis and myocardial infarction. Hepcidin measurement also may be useful in

anticipating consequences of iron supplementation in infectious and

inflammatory diseases.



Hepcidin in Human Iron Disorders



Iron-depleted HFE C282Y patients have been shown to have very low

serum hepcidin concentrations, reflecting the combined effects of the

HFE mutation and phlebotomy on hepcidin expression (125, 132). J Am Soc Mass Spectrom 2010;21:1515-25.



(53.) Vokurka M, Krijt J, Sulc K, Necas E. Circ J 2010; 74:301-6.



DISORDERS ASSOCIATED WITH HEPCIDIN EXCESS



(39.) Sow FB, Florence WC, Satoskar AR, Schlesinger LS, Zwilling

BS, Lafuse WP. ER stress-inducible factor CHOP affects the expression

of hepcidin by modulating C/EBPalpha activity. Under low iron conditions, membrane-bound HJV is cleaved by

matriptase-2, a transmembrane protease serine 6, encoded bythe

transmembrane protease, serine 6 (TMPRSS6) [6] gene, expressed

predominantly in the liver (48, 49). Plasma hepcidin is a modest predictor of dietary iron

bioavailability in humans, whereas oral iron loading, measured by

stable-isotope appearance curves, increases plasma hepcidin. In a pediatric study, no relationship was found

between Helicobacter pylori infection and hepcidin and between cytokine

and hepcidin concentrations, suggesting that H. Recently,

several (199-201) but not all (202) studies showed increased urinary

hepcidin concentrations after exercise. Tohoku J Exp Med 2009;218:93-8.



(133.) Swinkels DW, Fleming RE Novel observations in hereditary

hemochromatosis: potential implications for clinical strategies.

Haematologica 2011;96:485-8.



(162.) Ferrucci L, Semba RD, Guralnik JM, Ershler WB, Bandinelli S,

Patel KV, et al. Characterization of the transition-metal-binding properties of

hepcidin. The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin

activation by cleaving membrane hemojuvelin. Serum hepcidin-20 is elevated during the acute phase of myocardial

infarction. In anemia of multiple myeloma, hepcidin is induced by increased

bone morphogenetic protein 2. Immunochemical and

mass-spectrometry-based serum hepcidin assays for iron metabolism

disorders. In HD patients

on ESA, hepcidin did not predict which patients increase their

hemoblobin after iron loading (17). Pathways underlying iron accumulation in human nonalcoholic fatty

liver disease. Detection of serum hepcidin in renal failure and

inflammation by using ProteinChip System. Distinct requirements for Hfe in basal and induced hepcidin levels

in iron overload and inflammation. Hepatology 2011;53:1333-41.



Hepcidin in hereditary hemochromatosis (HH). Assessment of urinary concentrations of hepcidin provides

novel insight into disturbances in iron homeostasis during malarial

infection. Iron regulatory hormone hepcidin decreases in chronic

heart failure patients with anemia. These results suggest that

quantification of serum hepcidin concentrations could be a predictor for

the therapeutic effect of oral iron administration. We recently

assessed serum hepcidin reference intervals stratified by age and sex

stratified in a reference set selected from a large, well-phenotyped

sample of the general population of the Dutch city of Nijmegen (109). Gastroenterology 2007;132:294-300.



(35.) Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward

DM. In small

studies in humans the fractional excretion of hepcidin has been

calculated to be as low as 0%-5% (29, 30), either because it is

reabsorbed, similarly to other small peptides, or because it is not

freely filtered. Fax +31-24-3668754; e-mail

d.swinkels@labgk.umcn.nl.



(218.) Poli M, Girelli D, Campostrini N, Maccarinelli F, Finazzi D,

Luscieti S, et al. At the same time it

should be noted that the relevance of measuring hepcidin-25 instead of

total hepcidin for clinical decision-making has not been systematically

investigated. Currently available hepcidin methods.



MS Reference



HPLC/UPLC (a) MS/MS Kobold et al. Conversely, the increased hepcidin

concentrations seen with inflammation might increase the virulence of

bacteria that depend on macrophage iron for their proliferation within

these cells (e.g., Salmonella and Mycobacteria spp.). Diseased red blood cells topple iron

balance. Serum hepcidin: reference ranges and

biochemical correlates in the general population. These hepcidin peptides form a hairpin structure, with 4

intramolecular disulfide bridges (Fig. Accurate determination of hepcidin concentrations in serum and

urine will improve our understanding of iron metabolism disorders and

may provide a useful tool in the differential diagnosis and clinical

management of such diseases.



At present, there is no single laboratory test that can

definitively distinguish ACD from IDA (160, 163). In sickle

cell disease, patients demonstrate the additional confounding factor of

inflammation. Therefore, the diagnostic

toolbox for hepcidin peptide measurements in animal models should be

improved and extended (75, 219, 222). Development of reference methods and

reference materials. N Engl J

Med 2005;352:1011-23.



(185.) Divakaran V, Mehta S, Yao D, Hassan S, Simpson S, Wiegerinck

E, et al. Role of alcohol in the regulation of iron

metabolism. Blood 2008;112:4292-7.



(183.) Amato A, Santoro N, Calabro P, Grandone A, Swinkels DW,

Perrone L, Miraglia Del Giudice E. HIF antagonists

are used in cancer to inhibit tumor angiogenesis (210). J Hepatol 2010;53: 941-9.



(106.) Forest JC, Burtis CA. This lack of reabsorption

may play a role in several disorders of iron metabolism that are

associated with tubular dysfunction and increased concentrations ofurine

hepcidin, such as inflammation, iron overload, and malaria (33, 34).

Possible local tubular production of hepcidin must also be taken into

account in the interpretation of urine as a mirror of serum hepcidin

concentrations in such studies (25) as well as possible defective

tubular reabsorption of hepcidin. ER stress controls iron metabolism through induction

of hepcidin. Nat Med 2007;13:1020-1.



Preclinical and clinical studies have also shown that hepcidin

antagonists such as hepcidin antibodies, BMP antagonists, cytokine

receptor antibodies, and HIF stabilizers can decrease hepcidin

expression and reverse iron abnormalities (208, 211-218).



(141.) Kroot JJ, Laarakkers CM, Kemna EH, Biemond BJ, Swinkels DW.

Regulation of serum hepcidin levels in sickle cell disease.

Haematologica 2009;94:885-7.



(26.) Gnana-Prakasam JP, Martin PM, Mysona BA, Roon P, Smith SB,

Ganapathy V. Humanized antiinterleukin-6 receptor antibody treatment

of multicentric Castleman disease. Biometals 2006;19: 527-33.



[4] Nonstandard abbreviations: AMI, acute myocardial infarction;

ACD, anemia of chronic disease; CKD, chronic kidney disease; RE,

reticuloendothelial; Tf-[Fe.sub.2], transferrin bound iron; TfR,

transferrin receptor; HFE, hemochromatosis iron protein; ERK/MAPK,

extracellular signal-regulated kinases/mitogen-activated protein kinase;

BMP/SMAD, bone morphogenetic protein/mothers against decapentaplegic

homolog (Drosophila); HJV, hemojuvelin; ESA, erythropoiesis-stimulating

agent; EPO, erythropoietin; GDF15, growth differentiation factor-15;

TWSG1, twisted gastrulation protein homolog-1; HIF, hypoxia inducible

factor; DMT, divalent metal transporter; IL, interleukin; C/EBP,

CCAAT-enhancer-binding protein; c, competitive; MS, mass spectrometry;

IDA, iron deficiency anemia; HH, hereditary hemochromatosis; HD,

hemodialysis.



(204.) Nemeth E. Inhibitors of the key initiators of the

cellular hypoxic response, such as prolyl hydroxylases, prevent

inactivation and degradation of HIF and as such maybe effective hepcidin

suppressors and restore natural iron regulation in anemia (220, 221).



In several early studies a commercial serum-based immunoassay was

used that measures the hepcidin precursor prohepcidin rather than the

bioactive peptide (80). The peptide hormone hepcidin interacts with the cellular

iron exporter ferroportin and thus is now recognized as the key

regulator of systemic iron homeostasis. Genes

that are regulated through E-boxes, including the clock genes period

homolog 1 (Drosophila) (PER1), timeless homolog (Drosophila) (TIMELESS),

and clock homolog (mouse) (CLOCK), tend to be under circadian rhythmic

transcriptional control. Daily regulation of serum and urinary hepcidin is not

influenced by submaximal cycling exercise in humans with normal iron

metabolism. Cell 2010;142:24-38.



Received May 10, 2011; accepted July 20, 2011.



(70.) Harrison-Findik DD. It has been

hypothesized that increased hepcidin concentrations may enhance

cardiovascular risk by increasing intracellular macrophage iron

concentrations and by increasing their atherogenic potential (186-189).

Furthermore, recent studies showed increased hepcidin production in

patients with (low-grade) inflammation, obesity, and nonalcoholic fatty

liver disease, which are traditionally associated with cardiovascular

disease (21, 144, 190, 191). Blood 2005; 106:2627-32.



(137.) Origa R, Galanello R, Ganz T, Giagu N, Maccioni L, Faa G,

Nemeth E.

(124.) Lee PL, Beutler E. Therefore, a

second Round Robin for plasma hepcidin was initiated to explore

word-wide hepcidin harmonization by exchanging synthetic materials for

assay harmonization and native samples. This latter variation is not surprising given

the absence of a reference method (106) and a validated commutable calibrator or other material for assay harmonization. However, there are 2 classes of

ferroportin mutations with different phenotypic consequences: loss of

hepcidin regulation and loss of ferroportin function (36, 128-130). Eur J Appl Physiol 2005;95:569-71.



(67.) Vecchi C, Montosi G, Zhang K, Lamberti I, Duncan SA, Kaufman

RJ, Pietrangelo A. High-throughput protein characterization using

mass spectrometric immunoassay. The first assays to

measure bioactive hepcidin-25 were an immunodot assay (59), SELDI-TOF

mass spectrometry (MS) (12, 15, 84). Blood Cells Mol Dis

2005;35:177-81.



(16.) Peters HP, Laarakkers CM, Swinkels DW, Wetzels JF. Determinants of hepcidin in patients on maintenance

hemodialysis: role of inflammation. Urinary hepcidin: an

inverse biomarker of acute kidney injury after cardiopulmonary bypass?

Curr Opin Crit Care [Epub ahead of print 2010 Aug 21].



Serum hepcidin concentrations appear to be determined by the

relative strengths of the individual regulators. In conclusion, hepcidin is a

promising diagnostic tool but efforts must be undertaken to assess the

relevance of specifically measuring hepcidin-25, to harmonize assay

outcomes throughout the world, to define clinical decision limits, and

to make assays available to clinical laboratories before hepcidin assays

can be fully included in clinical practice.



Concluding Remarks



(215.) Hashizume M, Uchiyama Y, Horai N, Tomosugi N, Mihara M.

Tocilizumab, a humanized antiinterleukin-6 receptor antibody, improved

anemia in monkey arthritis by suppressing IL-6-induced hepcidin

production. Plasma hepcidin levels are elevated but responsive to

erythropoietin therapy in renal disease. PLoS ONE 2008;3: e2706.



(60.) Peyssonnaux C, Zinkernagel AS, Schuepbach RA, Rankin E,

Vaulont S, Haase VH, et al. falciparum and P. Clearly, future large and

well-designed studies are needed to improve our understanding of this

hepcidin regulatory network in various disorders of iron metabolism.



(118.) Cherian S, Forbes DA, Cook AG, Sanfilippo FM, Kemna EH,

Swinkels DW, Burgner DP. Decreased serum hepcidin and improved

functional iron status 6 months after restrictive bariatric surgery.

Obesity 2010;18:2010-6.



Author Contributions: All authors confirmed they have contributed

to the intellectual content of this paper and have met the following 3

requirements: (a) significant contributions to the conception and

design, acquisition of data, or analysis and interpretation of data; (b)

drafting or revising the article for intellectual content; and (c) final

approval of the published article.



Hepcidin in sports medicine. Time-course analysis of

serum hepcidin, iron and cytokines in a C282Y homozygous patient with

Schnitzler's syndrome treated with IL-1 receptor antagonist.

Haematologica 2009;94:1297-300.



(72.) Lasocki S, Millot S, Andrieu V, Letteron P, Pilard N, Muzeau

F, et al. Natural and synthetic STAT3 inhibitors reduce

hepcidin expres sion in differentiated mouse hepatocytes expressing the

active phosphorylated STAT3 form. J Hepatol 2008;48: 801-10.



(96.) Busbridge M, Griffiths C, Ashby D, Gale D, Jayantha A,

Sanwaiya A, Chapman RS. J Clin Invest 2007;117:1933-9.



(1.) Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG,

Schulz-Knappe P, Adermann K. The low hepcidin state

relative to ferritin in these patients (125-127) leads to increased

intestinal iron absorption and body iron overload. This cleavage bymatriptase-2

weakens the BMP signaling.



(199.) Roecker L, Meier-Buttermilch R, Brechtel L, Nemeth E, Ganz

T. Nephrol Dial Transplant 2010;25:3996-4002.



https://www.thefreelibrary.com/Hepcidininhumanirondisorders:diagnosticimplications.-a0324761278

MS assays require relatively expensive equipment, but they have the

advantage of distinguishing between hepcidin-25, -22, and -20 (13).

ELISA assays will measure total hepcidin concentrations, with (depending

on the specificity of the antibody) different contributions from each of

these 3 isoforms. (99)

c-RIA Bushbridge et al. Immunoassays have the potential for more widespread use in

clinical laboratories. N Engl J Med 2009;361:1904-6.



Hepcidin in iron-loading anemias. For example, the

accumulation of hepcidin-22 and -20 isoforms may raise questions

regarding the interpretation that measured hepicidin concentrations

reflected bioactive hepcidin-25 in studies in which certain immunoassays

were used. Am J Nephrol 2010;31: 534-40.



(81.) Kemna E, Pickkers P, Nemeth E, van der Hoeven H, Swinkels D.

Time-course analysis of hepcidin, serum iron, and plasma cytokine levels

in humans injected with LPS. et al. Under

physiological conditions hepcidin-20 and hepcidin-22 are present in the

urine, but not, or at very low concentrations, in the serum (11-13).

Interestingly, these smaller hepcidin isoforms occur only in serum of

patients with diseases that are associated with increased concentrations

of hepcidin-25, such as acute myocardial infarction (AMI), [4] sepsis,

anemia of chronic disease (ACD), metabolic syndrome, and chronic kidney

disease (CKD) (Fig. Blood 2007;110:1048-54.



(127.) Papanikolaou G, Tzilianos M, Christakis JI, Bogdanos D,

Tsimirika K, MacFarlane J, et al. However,

the optimal target concentration of hemoglobin is debated, and many

patients become ESA resistant. Arterioscler Thromb Vasc Biol 2011;31:683-90.



Effect of iron administration on hepcidin concentrations. Also, heparin and heparin derivatives have

recently been shown to inhibit hepcidin expression in vitro and in vivo

by interfering with BMP signaling (218).



(29.) Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M.

Immunoassay for human serum hepcidin. (13)

Swinkels et al. (30)

Anderson et al. Blood Cells Mol Dis

2009;43:169-79.



(28.) Peslova G, Petrak J, Kuzelova K, Hrdy I, Halada P, Kuchel PW,

et al. J Biol Chem 2002;277:37597-603.



(8.) Tselepis C, Ford SJ, McKie AT, Vogel W, Zoller H, Simpson RJ,

et al. Swinkels [1,2] *



Since the discovery of hepcidin 10 years ago, multiple studies have

contributed insights into the regulation of hepcidin and its functional

properties. Serum

hepcidin-25 levels in patients with chronic kidney disease are

independent of glomerular filtration rate. Expert Opin Ther Targets 2006;10:267-80.



In addition to its potential as a marker of iron metabolism in

patients with CKD, combined urinary and serum hepcidin concentrations

may serve as a marker for renal disease itself. Liver iron concentrations and urinary hepcidin in

beta-thalassemia. STAT3 is required for IL-6-gp130-dependent

activation of hepcidin in vivo. Training surface and intensity: inflammation,

hemolysis, and hepcidin expression. Br J Haematol 2009; 146:317-25.



In patients with heritable forms of anemia, information on hepcidin

concentrations may be valuable for identifying the most severely

affected patients and predicting and monitoring parenchymal iron

overload.



(150.) Hartman KR, Barker JA Microcytic anemia with iron

malabsorption: an inherited disorder of iron metabolism. Blood

2006;108:4237-45.



(98.) Koliaraki V, Marinou M, Vassilakopoulos TP, Vavourakis E,

Tsochatzis E, Pangalis GA, et al. The swinging pendulum of the anemia of cancer:

erythropoietin trumps hepcidin. Anal Biochem 2008;384:245-53.



(50.) Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont

S. To allow proper interpretation of the effect of the

erythropoietic drive on hepcidin production, EPO and hepcidin

concentrations should be assessed simultaneously in selected patients

with anemia of CKD.



(122.) van Bokhoven MA, van Deursen CT, Swinkels DW. Hepcidin mediates transcriptional changes that modulate acute

cytokine-induced inflammatory responses in mice. Eur J Haematol

2009;83:595-602.



(18.) Rivera S, Nemeth E, Gabayan V, Lopez MA, Farshidi D, Ganz T.

Synthetic hepcidin causes rapid dose-dependent hypoferremia and is

concentrated in ferroportin-containing organs. Several

studies have addressed the relationship between iron absorption and

hepcidin concentrations. The

N-terminus of hepcidin is essential for its interaction with

ferroportin: structure-function study. Furin-mediated release

of soluble hemojuvelin: a new link between hypoxia and iron homeostasis.

Blood 2008;111:924-31.



Stock Ownership: None declared.



(79.) Hunter HN, Fulton DB, Ganz T, Vogel HJ. Measurement of serum hepcidin may prove to be useful as a

marker of iron dysregulation in these forms of iron overload.



(211.) Sasu BJ, Cooke KS, Arvedson TL, Plewa C, Ellison AR, Sheng J, et al. (13) (Bachem)

Ganz et al. Hepcidin-a potential novel biomarker for iron status in

chronic kidney disease. 4).

Circulating transferrin appears to be sensed via a hepatocellular

complex, which includes transferrin receptor-1 (TfR1), TfR2, and

hemochromatosis iron protein (HFE). For instance, studies

suggest that smaller increases in urinary hepcidin, relative to serum

hepcidin, maybe associated with a greater risk of acute kidney injury

after coronary artery bypass graft surgery in patients with stable renal

function (178, 179).Ina study on urine biomarkers in patients with lupus

nephritis, urinary concentrations of hepcidin, possibly secreted by

interstitial inflammatory cells, were identified as markers of renal

lupus nephritis flare (108).



(19.) Nemeth E, Preza GC, Jung CL, Kaplan J, Waring AJ, Ganz T. Biochem J 2008;413:553-7.



(51.) Pinto JP, Ribeiro S, Pontes H, Thowfeequ S, Tosh D, Carvalho

F, Porto G. In an attempt to

delineate relative strengths of these regulators in patients with iron

metabolism disorders, we constructed an algorithm to predict relative

hepcidin concentrations based on certain serum parameters (C-reactive

protein, Tf saturation, and soluble TfR) that reflect inflammation,

serum iron, and erythropoietic regulatory pathways (71). Blood 2007;109: 353-8.



(37.) Delaby C, Pilard N, Goncalves AS, Beaumont C, Canonne-Hergaux

F. Intra-individual

variability in serum hepcidin precludes its use as a marker of iron

status in hemodialysis patients. Blood 2009;113:3593-9.



FIRST-GENERATION (SEMIQUANTITATIVE) HEPCIDIN ASSAYS



(105.) Kroot JJ, Kemna EH, Bansal SS, Busbridge M, Campostrini N,

Girelli D, et al. Rapid Commun

Mass Spectrom 2007;21:4033-8.



(89.) Li H, Rose MJ, Tran L, Zhang J, Miranda LP, James CA, Sasu

BJ. SELDI-TOF-MS determination of hepcidin in clinical samples

using stable isotope labelled hepcidin as an internal standard. Clin J Am Soc Nephrol

2009;4:1331-7.



Circulating hepcidin was recently found to be bound to

[[alpha].sub.2]-macroglobulin with relatively high affinity and to

albumin with relatively low affinity. Haematologica 2008;93:90-7.



(176.) Kato A, Tsuji T, Luo J, Sakao Y, Yasuda H, Hishida A.

Association of prohepcidin and hepcidin-25 with erythropoietin response

and ferritin in hemodialysis patients Am J Nephrol 2008;28:115-21.



(22.) Nguyen NB, Callaghan KD, Ghio AJ, Haile DJ, Yang F. In healthy human volunteers hepcidin in urine

and serum increased upon oral iron dosing, although nonresponders were

reported (29, 59, 112, 145-147). Clin Chem 2009;55:1354-60.



(207.) Gardenghi S, Ramos P, Marongiu MF, Melchiori L, Breda L, Guy

E, et al. Iron homeostasis in mother and child during placental

malaria infection. (100)



Hepcidin binding



Q-TOF LC-MS Crockett et al. Development of a novel immunoassay for the iron

regulatory peptide hepcidin. The effects of erythropoetic activity and iron

burden on hepcidin expression in patients with thalassemia major.

Haematologica 2006;91:809-12.



The activation of the hepcidin regulatory pathways described above

are all dependent on interactions of circulating or autocrine factors

with hepatocyte membrane sensors to control the hepatic production of

hepcidin for proper maintenance of systemic iron homeostasis [reviewed

in (44)]. Trans R Soc

Trop Med Hyg 2010; 104:175-84.



[FIGURE 2 OMITTED]



Hepcidin Kinetics

(193.) Sharma S, Nemeth E, Chen YH, Goodnough J, Huston A, Roodman

GD, et al. Harmonization of the methodologies, by

using reliable calibrators or other materials for harmonization, will

enable translational scientists as well as physicians in clinical

practice around the world to collectively define criteria for the use of

hepcidin assays in diagnosis, staging, monitoring, and assessing

treatment indications of iron disorders.



(76.) Roy CN, Custodio AO, de Graaf J, Schneider S, Akpan I,

Montross LK, et al. Blood 2010;115: 3817-26.



(68.) Oliveira SJ, Pinto JP, Picarote G, Costa VM, Carvalho F,

Rangel M, et al. A crucial element in the maintenance of

systemic iron homeostasis is effective communication between cells that

absorb iron from the diet (duodenal enterocytes), use iron (mainly

erythroid precursors), and store iron (hepatocytes and tissue

macrophages). Regulation of type II transmembrane serine proteinase TMPRSS6

by hypoxia-inducible factors: new link between hypoxia signalling and

iron homeostasis. J Clin Invest

2004;113:1271-6.



Hepcidin-Modulating Agents



(13.) Kroot JJC, Laarakkers CM, Geurts-Moespot AJ, Grebenchtchikov

N, Pickkers P, van Ede AE, et al. Hepcidin in iron overload disorders.

Blood 2005; 105:4103-5.



Hepcidin in renal diseases. Although in vitro

studies suggest bacteriocidal effects of hepcidin, these effects would

require concentrations higher than those observed in the circulation.

Such concentrations may be achieved locally, for instance in phagosomes

of infected macrophages (39). Erythropoiesis requires considerable amounts of

iron, so suppression of hepatic hepcidin synthesis by erythropoietic

signals is of great physiological importance. Kidney Int 2009;76:912-3.



(148.) Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA,

Hartman KR, et al. These differences

in level assignment hinder the comparability of data collected by use of

the various methods and preclude the definition of universal reference

intervals and cutoff values for clinical decision making. Am J Clin Nutr

2009;89:1088-91.



(145.) Lin L, Valore EV, Nemeth E, Goodnough JB, Gabayan V, Ganz T.

Iron transferrin regulates hepcidin synthesis in primary hepatocyte

culture through hemojuvelin and BMP2/4. However, there are

indirect mechanisms by which HIFs may regulate hepcidin expression.

Increased HIF activity is associated with increased matriptase-mediated

cleavage hemojuvelin and thus decreased hepcidin expression (62).

Moreover, the systemic iron regulation by the hepcidin-ferroportin axis

acts in harmony with the enterocyte cellular iron homeostasis control

system. Hepcidin as a therapeutic tool to limit iron overload and

improve anemia in beta-thalassemic mice. Endocrinology 2007;148: 2663-8.



Hepcidin in cancer. Science 2004;306: 2090-3.



(139.) Malcovati L, Porta MGD, Laarakkers CM, Galli A, Albertini R,

Maffioli M, et al. Blood 2007; 110:2182-9.



(146.) Young MF, Glahn RP, riza-Nieto M, Inglis J, Olbina G,

Westerman M, O'Brien KO. However, this concern might be overcome by exploiting

antibodies that are hepcidin-25 specific (100). These findings again suggest that

the erythroid demand for iron might be a more powerful regulator of

hepcidin expression than inflammatory- or iron-induced hepcidin

formation. Eur J Clin Invest 2009;39: 883-90.



Interestingly, hepcidin also was evaluated as a predictor of ESA

response in several relatively small studies in humans. Pediatr Nephrol 1998;12:280-3.



(69.) Bridle K, Cheung TK, Murphy T, Walters M, Anderson G,

Crawford DG, Fletcher LM. Science 2009;325:877-80.



(55.) Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee YT, et al.

High levels of GDF15 in thalassemia suppress expression of the iron

regulatory protein hepcidin. A

novel TMPRSS6 mutation that prevents protease auto-activation causes

IRIDA. Hepcidin

concentrations in untreated patients with rare juvenile forms of HH tend

to be very low (127). Proinflammatory state, hepcidin, and anemia in older

persons. The pathogenesis of septic

acute renal failure. Plasma hepcidin concentrations significantly predict interindividual

variation in iron absorption in healthy men. Hepcidin biology and therapeutic applications.

Expert Rev Hematol 2010;3:153-5.



REGULATION BY IRON STATUS



REGULATION BY HYPOXIA



(102.) Nicolardi S, Palmblad M, Dalebout H, Bladergroen M,

Tollenaar RA, Deelder AM, van der Burgt YE. HFE mutations modulate the effect of iron on serum

hepcidin-25 in chronic hemodialysis patients. Development of a method for the sensitive and quantitative

determination of hepcidin in human serum using LC-MS/MS. Clin Chem 2010;56:1570-9.



(201.) Peeling P, Dawson B, Goodman C, Landers G, Wiegerinck ET,

Swinkels DW, Trinder D. J Infect Dis 2009;199:253-62.



Small hepcidin peptides have been shown to act as agonists in mice

in vivo on the basis of their amino-terminal domain (206). 3).



(172.) Kuragano T, Shimonaka Y, Kida A, Furuta M, Nanami M, Otaki

Y, et al. Elevated growth differentiation factor 15 expression

in patients with congenital dyserythropoietic anemia type I. Blood

2005;106: 3268-70.



REGULATION BY ERYTHROPOIETIC SIGNALS



(6.) Gerardi G, Biasiotto G, Santambrogio P, Zanella I, Ingrassia

R, Corrado M, et al. The

consequences of ineffective erythropoiesis on hepcidin expression thus

appear to trump the consequences of excessive iron stores. The gene encoding the iron regulatory peptide hepcidin is

regulated by anemia, hypoxia, and inflammation. Notably, monitoring of the bioactive serum hepcidin

during treatment by these hepcidin antagonists appears to be complicated

by the interference of the compounds in the assays (219).



(123.) Swinkels DW, Janssen MC, Bergmans J, Marx JJ. Hepcidin might thus be a mediator of the high incidence of iron

deficiency among athletes.



(85.) Murphy AT, Witcher DR, Luan P, Wroblewski VJ. On the basis of some

studies, investigators have reported that hepcidin binds divalent metals

([Cu.sup.2+], [Fe.sup.2+], [Zn.sup.2+], and [Ni.sup.2+]), but these

findings are inconsistent, e.g., some studies have suggested the

presence of iron in the core of the peptide in a tetrahedral sulfur

coordination (4-6), whereas others provide evidence for an

amino-terminal Cu(2+)-Ni(2+)-binding (ATCUN) motif (7, 8). Subsequent amino-terminal processing of the 25 amino acid

form can result in the appearance of 2 smaller hepcidin forms of 22 and

20 amino acids. Overweight children

have higher circulating hepcidin concentrations and lower iron status

but have dietary iron intakes and bioavailability comparable with normal

weight children. (90)

Altamura et al. A

metal-binding site is present in the amino terminal region of the

bioactive iron regulator hepcidin-25. Hepcidin: a new tool in the

management of anaemia in patients with chronic kidney disease? Nephrol

Dial Transplant 2008;23:2450-3.



(114.) Rehu M, Punnonen K, Ostland V, Heinonen S, Westerman M,

Pulkki K, Sankilampi U. Serum and liver iron differently regulate the bone

morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice.

Hepatology 2011;54:273-84.



(36.) De Domenico I, Ward DM, Nemeth E, Vaughn MB, Musci G, Ganz T,

Kaplan J. A time course of hepcidin response to iron challenge

in patients with HFE and TfR2 haemochromatosis. Nephrol Dial Transplant

2010;25:848-53.



(95.) Ward DG, Roberts K, Stonelake P, Goon P, Zampronio CG, Martin

A, et al. Blood 2010;116: 3635-44.



(83.) Roe MA, Spinks C, Heath AL, Harvey LJ, Foxall R, Wimperis J,

et al. Hepcidin, the iron watcher. Other studies have shown

that increased and/or ineffective erythropoiesis blunts the induction of

hepcidin by increased body iron stores (52, 73). As mentioned earlier, several

investigators have reported a decrease in hepcidin concentrations after

EPO administration (31, 50, 169, 170). (95)



Immunochemical



c-ELISA Kroot et al. Am J Hematol

1996;51:269-75.



(175.) Ford BA, Eby CS, Scott MG, Coyne DW. Am J Clin Nutr 2008;87:1374-83.



Hepcidin clearance is assumed to occur via cellular codegradation

with ferroportin at its sites of action, and via excretion by the

kidneys. Free Radic Biol Med 2007;42:492-8.



Although hepcidin is a promising companion diagnostic for ESA

therapy, patients with renal insufficiency are a rather complex

population in which consistent results have been difficult to obtain

(175). (85)

Murao et al. Clin

Chem 2006;52:950-68.



(156.) Nweneka CV, Doherty CP, Cox S, Prentice A. Because of its low molecular weight and small radius, unbound hepcidin is likely to freely pass into the glomerular filtrate. Metabolism

2005;54:453-9.



(159.) Drakesmith H, Prentice A. Cis and trans regulation of

hepcidin expression by upstream stimulatory factor. Most promising applications of serum

hepcidin in diagnostic medicine.



Disease condition Expected hepcidin

concentrations (a)



Classical Low, maybe compensated

hereditary by iron overload over

hemochromatosis time



Iron-loading Low, maybe compensated

anemias by transfusional iron

overload over time



Acquired forms of Miscellaneous

iron overload



Iron-refractory Inappropriately high

iron deficiency

anemia

Inflammation and High

infection



Chronic kidney High, decreases upon

diseases EPO treatment



Acute kidney Low (urine)

injury

Treatment with Depends on the disorder

hepcidin

antagonists and

agonists



Disease condition Potential added value

of hepcidin diagnostics



Classical Screen for the presence

hereditary of hemochromatosis

hemochromatosis

Predict which homozygotes

will be at risk for iron

overload



Determine the phlebotomy

interval



Prioritize genes to be

investigated

Iron-loading Identify the most

anemias severely affected

patients



Predict and monitor

(parenchymal) iron

overload



Acquired forms of A marker of iron

iron overload dysregulation



Iron-refractory Screen for primary defect

iron deficiency in hepcidin regulation

anemia

Inflammation and Differentiate ACD (b) and

infection IDA



Guide iron supplementation

therapy

Chronic kidney Predict EPO response

diseases

Guide treatment with EPO,

intravenous iron

Acute kidney Diagnose acute kidney

injury injury

Treatment with Monitor and assess

hepcidin indications

antagonists and

agonists



(a) In serum, unless stated otherwise.



(b) ACD, anemia of chronic disease; IDA, iron

deficiency anemia; EPO, erythropoietin.

(213.) Fatih N, Camberlein E, Island ML, Corlu A, Abgueguen E,

Detivaud L, et al. Int J Lab Hematol

2010;33:187-93.



(71.) Kemna EH, Kartikasari AE, van Tits LJ, Pickkers P, Tjalsma H,

Swinkels DW. Hepcidin, the hormone of iron metabolism, is bound specifically

to alpha-2-macroglobulin in blood. if you are handy you can probably replace the battery and or alternator in your car. The problem is that the problem with your vehicle might not be caused by the battery or the alternator! It might be a short in a wire, a bad ground or just a sticky relay that causes the battery to drain or the alternator to not charge the battery. That is why there are professional auto repair shops! http://www.ikev.org/haber/

stabilite/cd/08%201.7%20ICH%20Q2A%20for %20CD.pdf (Accessed June 2011).



REFERENCE INTERVALS FOR SERUM HEPCIDIN



(92.) Crockett DK, Kushnir MM, Phillips JD, Rockwood AL.

Time-of-flight mass spectrometry analysis of the ferroportin-hepcidin

binding domain complex for accurate mass confirmation of bioactive

hepcidin 25. Anal Biochem 2009;389:124-9.



Interpretation of hepcidin test results might be influenced by

whether the various assays are detecting freely circulating hepcidin,

hepcidin bound to a carrier protein, or both of these hepcidin species.

Lastly, the presence of hepcidin-22 and -20 isoforms, which play no role

in the regulation of iron metabolism, can interfere with the

quantification of hepcidin-25 in immunoassays that use antibodies that

react with all of these hepcidin isoforms.



(5.) Farnaud S, Rapisarda C, Bui T, Drake A, Cammack R, Evans RW.

Identification of an iron-hepcidin complex. Eur J Appl

Physiol 2009;106:51-9.



(138.) Tamary H, Shalev H, Perez-Vraham G, Zoldan M, Levi I,

Swinkels DW, et al. GDF15 and TWSG1 are

both BMP antagonists produced by erythroblasts and putative components

of the erythroid regulator that can inhibit hepcidin expression in

vitro. Gut

2004;53:735-43.



(112.) Roe MA, Collings R, Dainty JR, Swinkels DW, Fairweather-Tait

SJ. Furthermore, as the field moves toward

the use of hepcidin-related therapies, it should be realized that the

outcome of assays that are used to monitor the fate of hepcidin during

antihepcidin therapy may be affected by compounds that interfere with

hepcidin. The iron-regulatory peptide hormone hepcidin:

expression and cellular localization in the mammalian kidney. The

results of this study suggest that administration of antihepcidin

therapies alone or in combination with erythropoiesis-stimulating agents

may improve patients' erythropoietic response and allow the use of

lower EPO doses to avoid the potential detrimental effects of high EPO

concentrations. Most patients with iron-refractory

IDA are resistant to dietary iron supplementation, owing to a defect in

the TMPRSS6 gene encoding matriptase-2. Blood 2005;106: 1864-6.



(210.) Semenza GL. The ability

of hepcidin to bind iron and other divalent metals suggests there may be

a nonhormonal role for hepcidin in iron metabolism or a conformational

mechanism for uptake of divalent metals as part of hepcidin's

hormonal role in regulating ferroportin degradation.



(14.) Suzuki H, Toba K, Kato K, Ozawa T, Tomosugi N, Higuchi M, et

al. The hepcidin/ferritin ratio is also found to be

low in patients with these disorders (135, 137, 138, 140-142). The microcytic red cell and the anemia

of inflammation. Identification of TWSG1 as a second novel

erythroid regulator of hepcidin expression in murine and human cells.

Blood 2009;114:181-6.



Employment or Leadership: H. Infections are

associated with marked changes in iron homeostasis (152).

Malaria-associated anemia has been found to be associated with low serum

iron and increased hepcidin concentrations, causing iron withholding in

the RE system that ultimately leads to iron-restricted erythropoiesis

(115, 118, 153-156). J Endocrinol 2008;197:241-9.



(180.) Tussing-Humphreys LM, Nemeth E, Fantuzzi G, Freels S, Guzman

G, Holterman AX, Braunschweig C. Hepcidin expression in mouse retina and its regulation via

lipopolysaccharide/Toll-like receptor-4 pathway independent of Hfe.

Biochem J 2008;411:79-88.



Acknowledgments: We thank Mirian Janssen and Paul Brons and the

members of the Dutch HEPCIKID (HEPCIdin in patients with KIdney Disease)

consortium, Jack Wetzels, Carlo Gaillard, Roos Masereeuw, Jaap Joles,

and Hilde Peters, for useful discussions on the diagnostic potential for

hepcidin measurements in clinical practice; Fred Sweep, Alec Ross, Guus

Kortman, Rieneke Terink, Susanne van Santen, Lucas van Eijk, Tessel

Galesloot, Louise de Swart, Charlotte Schaap, Erwin Wiegerinck, Coby

Laarakkers, Siem Klaver, Rian Roelofs, Anneke Geurts-Moespot, Nicolai

Grebenchtchikov, and other members of the Nijmegen Iron Group for

stimulating discussions on hepcidin methodology and its clinical

utility; Hans Willems for discussion on assay harmonization; and Hanka

Venselaar and Erwin Kemna for their contribution to the figures.



(140.) Casanovas G, Swinkels DW, Altamura S, Schwarz K, Laarakkers

CM, Gross HJ, et al. Anemia of inflammation: the cytokine-hepcidin

link. Blood

2009;113:5277-86.



The functional signaling routes by which (a) iron status, (b)

erythropoietic activity, (c) hypoxia, and (d) inflammation affect

hepcidin expression are increasingly being investigated. ASH Annu

Meeting Abstracts 2008;112:2676.



(62.) Lakhal S, Schoedel J, Townsend AR, Pugh CW, Ratcliffe PJ,

Mole DR. A decrease in hepcidin results in

the release of stored iron and an increase in dietary iron absorption.

On the other hand, infection and inflammation cause an increase in

hepcidin synthesis. Thus, in chronic mild inflammatory

conditions, such as obesity or the metabolic syndrome, even a mild

hepcidin excess may be sufficient to alter the balance between iron loss

and iron uptake toward iron deficiency.



Much is still unknown on the origin of the smaller isoforms of

hepcidin, although data suggest that calcium-independent tissue activity

in pancreas extracts might lead to the systemic N-terminal truncation of

hepcidin-25 to hepcidin-22, and that dipeptidylpeptidase 4 is involved

in the processing of hepcidin-22 into hepcidin-20 (9, 10). J

Biol Chem 2009;284:24155-67.



In ACD, IL-6 and other cytokines induce hepcidin concentrations,

resulting in iron sequestration in macrophages and leading to

hypoferremia. Nat Rev Microbiol 2008;6: 541-52.



(220.) Braliou GG, Verga Falzacappa MV, Chachami G, Casanovas G,

Muckenthaler MU, Simos G. Results of the first international round robin for the

quantification of urinary and plasma hepcidin assays: need for

standardization. These promises brought about a large series of

studies that examined hepcidin concentrations in patients with CKD. Soon after its discovery and during the

years thereafter, hepcidin was recognized as potentially relevant in CKD

because it may be responsible for the often-observed imbalance between

iron homeostasis and ESA resistance (50, 166). This apparent circadian rhythm of hepcidin

concentrations may be regulated by transcription factors such as

upstream stimulatory factor and c-Myc/Max through E-boxes (110). Blood

2006;108:3730-5.



(47.) Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA,

et al. Some of these studies also demonstrated correlations

between hepcidin and iron or transferrin saturation (169, 170). Subsequently, iron availability for the erythron becomes

limiting and contributes to the development of ACD (59, 75, 81, 160,

161). Haematologica 2007;92:583-8



Decreased hepcidin expression has been reported in response to

hypoxia in vivo (58, 59). Increased adipose tissue expression of hepcidin in severe obesity is

independent from diabetes and NASH. Is

hepcidin-25 a clinically relevant parameter for the iron status in

hemodialysis patients? J Ren Nutr 2010;20:S77-83.



[FIGURE 3 OMITTED]



(15.) Tomosugi N, Kawabata H, Wakatabe R, Higuchi M, Yamaya H,

Umehara H, Ishikawa I. J Biomed

Biotechnol 2010;2010:329646.



Hepcidin in iron-refractory IDA. This algorithm

provided insight into the interrelation of these pathways by showing

that hepcidin inhibition by erythropoiesis strongly interferes with

hepcidin upregulation by iron and that inflammation strongly increases

hepcidin regardless of iron status and erythropoietic activity. IL-6 mediates hypoferremia of inflammation by inducing the

synthesis of the iron regulatory hormone hepcidin. In these

conditions, dysregulation of hepcidin synthesis as a consequence of

reactive oxygen species, endoplasmic reticulum stress, and cytokine tumor necrosis factor-[alpha]-mediated pathways may contribute to

relatively decreased hepcidin synthesis and iron accumulation (59,

67-70, 144). However, how

erythropoiesis regulates hepcidin is not clear yet. In

addition, hepcidin's potential to become a target of treatment was

quickly acknowledged. Anti-interleukin 6 receptor antibody tocilizumab reduces the

level of serum hepcidin in patients with multicentric Castleman's

disease. These

molecules, like BMPs are members of the transforming growth

factor-[beta] family and thus possibly act through effects on the

BMP/SMAD pathway (Fig. Serum hepcidin concentrations decreased, and an increase

in iron status and intestinal absorption was observed in individuals

with weight loss (182, 183). Blood

2005;106:1092-7.



(43.) De Domenico I, Nemeth E, Nelson JM, Phillips JD, Ajioka RS,

Kay MS, et al. However, MS methods can also be

automated (101, 102), and would have to be optimized for hepcidin

because its amphiphatic character makes it readily stick to laboratory

plastics, especially when relatively small sample volumes are used in

large tubes.



(42.) Keel SB, Abkowitz JL. 4).



(10.) Valore EV, Ganz T. Blood 2010;115:3810-6.



Further comparison of the analytical characteristics of various

published assays is incomplete, because not all studies have reported

these characteristics in accordance with the STARD (STAndards for the

Reporting of Diagnostic accuracy studies) procedures (103) or validated

the reported assays according to recommendations of the International

Conference of the Harmonization (ICH) of analytical procedures (104).

Nonetheless, the scientific and medical community was informed on the

status and agreement of the current hepcidin methods by a recent

international send-out of samples for urine and serum hepcidin

measurement in 2009, the so-called "Round Robin 1." In this

study the correlations between the participating methods were generally

high and the between-sample and analytical variation of most methods

were similar (105). (97)

Sandwich ELISA Butterfield et al. Moreover, hepcidin concentrations in these patients

were associated with increased concentrations of monocyte

chemoattractantprotein-1 and vascular damage. Blood Cells Mol Dis 2010;44:16-21.



(80.) Kulaksiz H, Gehrke SG, Janetzko A, Rost D, Bruckner T,

Kallinowski B, Stremmel W. Autocrine formation of hepcidin induces iron retention in human

monocytes. 1), and the high degree of conservation of

hepcidin among a wide range of species, which diminishes the elicitation

of an immune response in host animals (50).



(212.) Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng

SA, et al. Furthermore, sample throughput is likely to be

higher for ELISA than for MS assays. Quantitation of hepcidin in serum using

ultra-high-pressure liquid chromatography and a linear ion trap mass

spectrometer. There are multiple potential opportunities for cross

communication among these pathways, and experimental evidence shows that

these pathways do not function completely independently of each other.



Anti-IL-6-receptor antibody (tocilizumab) was shown to suppress

IL-6-induced hepcidin production in Castleman disease (a rare disorders

with systematic inflammation and anemia), and it improved anemia in

arthritic monkeys (214-217). 4) (55-57). Eur J Appl Physiol 2010;110: 877-83.



(167.) Viatte L, Vaulont S. Possibly, these increased hepcidin concentrations

might be advantageous in the protection against extracellular growing

microorganisms by decreasing the extracellular supply of iron. 3) [reviewed in (44)]. Best

Pract Res Clin Gastroenterol 2009;23:171-83.



(86.) Murao N, Ishigai M, Yasuno H, Shimonaka Y, Aso Y. Clin Chem

2008;54:1584-6.



(191.) Aigner E, Theurl I, Theurl M, Lederer D, Haufe H, Dietze O,

et al. Br J Biomed Sci 2009; 66:150-7.



SECOND-GENERATION (QUANTITATIVE) HEPCIDIN ASSAYS



(94.) Altamura S, Kiss J, Blattmann C, Gilles W, Muckenthaler MU.

SELDI-TOF MS detection of urinary hepcidin. However, because the defect is downstream of

hepcidin, these patients do not have low hepcidin concentrations. Biochem J 2010;427:289-96.



(111.) Bozzini C, Campostrini N, Trombini P, Nemeth E, Castagna A,

Tenuti I, et al. As such, we recommend standardization of

sampling time to minimize within-individual and (pre)analytical

variability in clinical studies exploiting hepcidin measurements.



Hepcidin in IDA. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Am

J Kidney Dis 2009;53: 584-95.



Authors' Disclosures or Potential Conflicts of Interest: Upon

manuscript submission, all authors completed the Disclosures of

Potential Conflict of Interest form. Haematologica

2010;95:1660-7.



(143.) Porter JB. J Nutr

Biochem 2010;21:749-56.



(84.) Kemna E, Tjalsma H, Laarakkers C, Nemeth E, Willems H,

Swinkels D. neutralized hepcidin by

a monoclonal antibody in the Brucella abortus mouse model of anemia of

inflammation and found it to restore responsiveness to EPO (211). However, in this study increased hepcidin

concentrations appeared insufficient for the development of anemia

(195), suggesting that compensation by increased erythropoiesis through

increased EPO production may explain the lack of anemia in cancer

despite increased hepcidin concentrations (196).



Hepcidin in heart diseases. Clin Chem 2003;49:19-20.



(38.) Ramey G, Deschemin JC, Durel B, Canonne-Hergaux F, Nicolas G,

Vaulont S. Eur J Appl Physiol 2009;106:435-43.



(216.) Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M,

Nakano S, et al. Maternal serum hepcidin is low at term and

independent of cord blood iron status. 4). Hereditary

hemochromatosis due to resistance to hepcidin: high hepcidin

concentrations in a family with C326S ferroportin mutation. Iron and its relation to immunity and

infectious disease. J Pharmacol

Toxicol Methods 2009;59:171-80.



(135.) Gardenghi S, Marongiu MF, Ramos P, Guy E, Breda L, Chadburn

A, et al. Alternatively, the otherwise expected increased

circulating concentrations in patients with decreased renal filtration

may be offset by a compensatory feedback decrease in hepatic hepcidin

production.



The increase in hepcidin expression with inflammation is primarily

mediated by interleukin-6 (IL-6). Eur J Appl Physiol 2008;103:

381-91.



(4.) Farnaud S, Patel A, Evans RW. Gastroenterology 2006;131:788-96.



(184.) Matsumoto M, Tsujino T, Lee-Kawabata M, Naito Y, Akahori H,

Sakoda T, et al. The solution

structure of human hepcidin, a peptide hormone with antimicrobial

activity that is involved in iron uptake and hereditary hemochro

matosis. Int J Obes

2010;34:1772-4.





(125.) van Dijk BA, Laarakkers CM, Klaver SM, Jacobs EM, van Tits

LJ, Janssen MC, Swinkels DW. Lancet 2006;367:133-43.



(128.) Sham RL, Phatak PD, Nemeth E, GanzT. One study

revealed that hepcidin concentrations of EPO responders did not differ

from those of hyporesponders (176), whereas cross-sectional studies

among HD patients and a prospective study among patients with combined

CKD and chronic heart failure revealed that nonresponders had low

hepcidin concentrations (32, 170, 177). Hepcidin is not useful as a biomarker for iron needs

in haemodialysis patients on maintenance erythropoiesis-stimulating

agents. LEAP-1, a novel highly disulfide-bonded

human peptide, exhibits antimicrobial activity. Future studies are needed to

assess the position of hepcidin in clinical decision making in these

patients.



(174.) Zaritsky J, Young B, Gales B, Wang HJ, Rastogi A, Westerman

M, et al. Iron absorption in dysmetabolic iron overload syndrome is

decreased and correlates with increased plasma hepcidin. J Clin Invest 2004;113: 1251-3.



Hepcidin Function



(206.) Nemeth E. Hepcidin in the management of

patients with mild nonhemochromatotic iron overload: fact or fiction? J

Hepatol 2008;49:680-5.



Hepcidin measurements appear to have potential utility in

diagnostic algorithms to distinguish IDA from ACD and the combined

presence of IDA/ACD. Blood 2010;

116:3627-34.



(46.) Corradini E, Meynard D, Wu Q, Chen S, Ventura P, Pietrangelo

A, Babitt JL. J Clin Invest

2010;120:2395-405.



(151.) Beutler E, Van GC, te Loo DM, Gelbart T, Crain K, Truksa J,

Lee PL. Hepcidin in anemia of chronic heart failure. Measurement of urinary hepcidin levels by SELDI-TOF-MS

in HFE hemochromatosis. The hepcidin produced by these cells, however, is unlikely to

make a significant contribution to systemic circulating concentrations,

but may exert local effects in these tissues (see below).



(66.) Pietrangelo A, Dierssen U, Valli L, Garuti C, Rump A,

Corradini E, et al. Am J

Clin Nutr 2010; 92:1385-92.



Hepcidin produced by various cell types other than hepatocytes (see

above) may have local effects in these tissues. Br J Haematol 2008;142:979-85.



(30.) Swinkels DW, Girelli D, Laarakkers C, Kroot J, Campostrini N,

Kemna EH, Tjalsma H. In this study serum hepcidin was strongly associated with serum

ferritin in men and women, confirming findings of smaller previous

studies (29, 30, 107, 111, 112). Down-regulation of hepcidin resulting from long-term

treatment with an anti-IL-6 receptor antibody (tocilizumab) improves

anemia of inflammation in multicentric Castleman disease. Hepcidin mRNA levels in

mouse liver respond to inhibition of erythropoiesis. Impaired intestinal iron absorption in Crohn's

disease correlates with disease activity and markers of inflammation.

Inflamm Bowel Dis 2006;12: 1101-6.



Since the discovery of hepcidin, there has been a substantial

interest in developing a reliable assay of the peptide hormone in body

fluids. A novel immunological assay for

hepcidin quantification in human serum. Blood 2006;108:1381-7.



Hepcidin-25 is thought to be the major regulator of dietary iron

absorption and cellular iron release. Br J Nutr 2007;97:544-9.



(104.) ICH Steering Committee. Biochimie 2009;91:1335-8.



DISORDERS ASSOCIATED WITH HEPCIDIN DEFICIENCY



(73.) Huang H, Constante M, Layoun A, Santos MM. In patients with hepatitis C virus,

increased transferrin saturation and increased concentrations of serum

ferritin and hepatic iron have been observed. These

findings are corroborated by in vitro studies that showed that the

truncated 22 amino acid and 20 amino acid forms have greatly diminished

and almost complete loss of ferroportin regulatory activity,

respectively, compared with 25 amino acid hepcidin (19).



In addition to its role in regulating systemic iron metabolism,

hepcidin may also contribute to host defense. After

treatment with phlebotomy that normalized iron stores (as assessed by

serum ferritin), these patients have been found to have serum hepcidin

concentrations or serum hepcidin/ferritin ratios within or above

reference intervals (128, 131). Effects of routine



(2.) Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot

P, Loreal O. The

low-grade inflammation in obese and dysmetabolic iron overload syndrome

patients, (characterized by the association of increased body iron

stores and metabolic features), is associated with increased hepcidin

concentrations, leading to poor iron absorption and causing ACD (116,

117, 180, 181). Plos One 2009;4: e4581.



(75.) Theurl I, Aigner E, Theurl M, Nairz M, Seifert M, Schroll A,

et al. In congenital iron-loading

anemias, such as [beta]-thalassemia (major and intermedia) and

congenital dyserythropoietic anemia I and II, but also in acquired forms

such as myelodys-plastic syndrome types RA (refractory anemia) and RARS (RA with ringed sideroblasts), the diseased erythron dysregulates iron

homeostasis by inhibiting hepcidin synthesis, even in the presence of

iron overload (134-140). Iron-regulatory protein hepcidin is increased in female athletes

after a marathon. Serum

pro-hepcidin: measuring active hepcidin or a non-functional precursor?

Gut 2005;54: 169-70.



Research Funding: None declared.



References



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Rametta R, et al. Am J Clin Nutr 2009; 89:533-8.



As we discussed previously, basal hepcidin concentrations greatly

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[6] Human genes: TMPRSS6, transmembrane protease, serine 6; HFE,

hemochromatosis; PER1, period homolog 1 (Drosophila); TIMELESS, timeless

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IL-6-independent pathway may be attributed to increased BMP-2

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Biomarkers of lupus nephritis determined by serial urine proteomics.

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In recent years, substantial progress has been made by the

introduction of an internal standard to quantify hepcidin-25 in serum

and urine by an updated TOF-MS assay (13, 30). Ineffective erythropoiesis in beta-thalassemia is

characterized by increased iron absorption mediated by downregulation of

hepcidin and up-regulation of ferroportin. One study showed a blunted hepcidin response to

inflammatory stimuli in hemochromatosis (HFE) knock-out mice (76),

whereas results of other studies of the same mice or a patient with an

HFE mutation suggest that inflammatory induction of hepcidin is

independent from the HFE protein (77, 78). Additional studies to investigate the

possibility that local urinary infections or tubular dysfunction can

contribute to hepcidinuria are warranted.



(32.) Costa E, Swinkels DW, Laarakkers CM, Rocha-Pereira P, Rocha

S, Reis F. Cellular iron is mostly bound

within iron protoporphyrin (heme) and iron--sulfur clusters, which serve

as enzyme cofactors, or stored within the core of ferritin multimers.

Without adequate iron, cells lose their capacity for electron transport and energy metabolism. (Pre) analytical imprecision, between-subject

variability, and daily variations in serum and urine hepcidin:

implications for clinical studies. Blood 2007;109:5027-35.



(129.) Drakesmith H, Schimanski LM, Ormerod E, Merryweather-Clarke

AT, Viprakasit V, Edwards JP, et al. The first reliable assays to quantify hepcidin in human body

fluids have recently been developed, and proof-of-principle studies in

human iron disorders highlight hepcidin as a promising novel tool in

diagnostic medicine, as outlined in Table 2. As such hepcidin was

described as a promising companion diagnostic marker to predict ESA

responsiveness, and to guide treatment with ESA and intravenous iron. We

conclude by highlighting the most promising applications of hepcidin

assays in diagnostic medicine.

Preclinical studies and some clinical studies have provided

proof-of-concept for synthetic hepcidin-25, small hepcidin peptides, BMP

agonists, and HIF stabilizers to prevent iron overload attributed to

hepcidin deficiency.



(24.) Kulaksiz H, Fein E, Redecker P, Stremmel W, Adler G, Cetin Y.

Pancreatic beta-cells express hepcidin, an iron-uptake regulatory

peptide. Clin J Am Soc Nephrol 2010;5: 1010-4.



(110.) Bayele HK, McArdle H, Srai SK. Moreover, hepcidin was found to modulate

lipopolysaccharide-induced transcription in both cultured macrophages

and in vivo mouse models (40). 2-Oxoglutarate-dependent oxygenases control

hepcidin gene expression. Heparin: a potent inhibitor of hepcidin expression in

vitro and in vivo. Regulation of hepcidin: Insights from biochemical analyses

on human serum samples. Therefore, there is need

for further studies on serum hepcidin concentrations in larger and

well-designed studies to resolve the aforementioned discrepancies in the

literature.



(158.) Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ,

Dutta A, et al. Development of novel therapeutic strategies that

target HIF-1. Eur J Heart Fail

2010;12:943-50.





Urine hepcidin measurements may be attractive, especially for use

in research and measurements in children and patients in the

underdeveloped world, because this matrix allows noninvasive sampling.

Nevertheless, although a significant relationship was found between the

hepcidin concentrations of urine-serum sample pairs from healthy

controls and patients with disorders of iron metabolism (12, 29, 107),

urine hepcidin concentrations may not always accurately reflect serum

hepcidin concentrations. Primaire hemochromatose door

ferroportinegenmutaties: is er een plaats voor hepcidine in de

diagnostiek? Ned Tijdschr Klin Chem Labgeneesk 2011;36:6-11.



HEPCIDIN ANTAGONISTS



(103.) Bruns DE. Hepcidin, a urinary

antimicrobial peptide synthesized in the liver. Supraphysiological doses of exogenous BMP-6 for 10 days also were

reported to improve hepcidin deficiency, reduce serum iron, and

redistribute tissue iron to appropriate storage sites (RE macrophages,

spleen) in a mouse model for HFE hemochromatosis (209). Furthermore, CRP and IL-6 were shown

to be less relevant predictors in the setting of renal insufficiency

(16,170,172-175). Of the currently

available commercial immunochemical research kits for serum hepcidin, we

found the RIA and enzyme-immunoassay kits of Bachem (purchased November

2009 and August 2010, respectively) to be suitable to differentiate

between hepcidin concentrations in serum samples of controls and

patients with various iron disorders, whereas the bioactive hepcidin kit

of DRG Instruments, (purchased October 2009) gave similar concentrations

for all samples and could not discriminate between iron metabolism

disorders (unpublished results).



(195.) Hohaus S, Massini G, Giachelia M, Vannata B, Bozzoli V,

Cuccaro A, et al. However, absolute hepcidin concentrations differed

widely between the assays. Proteome Sci 2008;6:28.



CHALLENGES FOR HEPCIDIN ASSAYS



(117.) Aeberli I, Hurrell RF, Zimmermann MB. Elevated systemic hepcidin and iron

depletion in obese premenopausal females. Correlations between the expression

of TWSG1 expression and serum iron parameters and serum hepcidin

concentrations have not yet been determined in humans. Suppression of

hepcidin during anemia requires erythropoietic activity. Evidence for distinct pathways of hepcidin regulation by acute

and chronic iron loading in mice. Regulation of iron homeostasis in anemia of chronic disease and

iron deficiency anemia: diagnostic and therapeutic implications. Hepcidin: a putative iron-regulatory

hormone relevant to hereditary hemochromatosis and the anemia of chronic

disease. Eur J Haematol 2010; 85:345-52.



prophylactic supplementation with iron and folic acid on admission

to hospital and mortalityin preschool children in a high malaria

transmission setting: community-based, randomised, placebo-controlled

trial. (98)

Schwarz et al. An Hfe-dependent pathway mediates hyposideremia in

response to lipopolysaccharide-induced inflammation in mice. BMP-2 administration in mice was found to increase

liver hepcidin expression and reduce serum iron concentrations in vivo

(208). Clin J Am Soc Nephrol 2009;4:1051-6.



(208.) Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY.

Modulation of bone morphogenetic protein signaling in vivo regulates

systemic iron balance. Advances in quantitative hepcidin measurements by

time-of-flight mass spectrometry. Because these patients tend to be anemic, even a mild

hepcidin excess can be considered to be inappropriately high for the

level of anemia. A novel mono clonal antibody immunoassay for the

detection of human serum hepcidin. Blood 2006;108:3204-9.



There is an increasing demand for preclinical tools to assess the

efficacy of hepcidin-related therapies. Adipose tissue is an active

endocrine organ that releases several cytokines and adipokines, which

can contribute to the development of a low-grade systemic inflammation.

Furthermore, adipose tissue also can produce hepcidin (21). BMJ 2011;342:c7251.



Iron is required for the function of oxygen-binding molecules,

mainly hemoglobin and myoglobin, and iron-containing enzymes, including

the cytochrome system in mitochondria. Hepcidin is down-regulated in alcoholic liver

injury: implications for the pathogenesis of alcoholic liver disease.

Alcohol Clin Exp Res 2006;30: 106-12.



(3.) Jordan JB, Poppe L, Haniu M, Arvedson T, Syed R, Li V, et al.

Hepcidin revisited, disulfide connectivity, dynamics, and structure. This

situation suggests that reference intervals may have limitations when

used for the interpretation of individual hepcidin concentrations. Antihepcidin antibody treatment modulates iron metabolism and

is effective in a mouse model of inflammation-induced anemia. Hepcidin and iron-related gene expression in

subjects with Dysmetabolic Hepatic Iron Overload. The authors concluded that hepcidin-20, rather than

hepcidin-25, may be helpful for the diagnosis of acute myocardial

infarction (14).



(99.) Schwarz P, Strnad P, von FG, Janetzko A, Krayenbuhl P, Adler

G, Kulaksiz H. Quantification of hepcidin-25 in human serum by

isotope dilution micro-HPLC-tandem mass spectrometry. Haematologica 2009; 94:1748-52.



(181.) Ruivard M, Laine F, Ganz T, Olbina G, Westerman M, Nemeth E,

et al. Therefore,

differentiation between these conditions is often achieved by combining

various biochemical markers of iron metabolism. http://www.ifcc.org/

index.asp?cat=Publications&scat=eJIFCC_&suba =

VoL13_No_3&subx=Development_Of_Reference_

Methcds_And_Reference_Materials_byJean-Claude_

Forest_Cari_A_Burtis&zip=1&dove=1&zona=full&

numero=&aq=1 (Accessed October 2011).



Hepcidin concentrations can also be induced by targeting the BMP

pathway (Fig. (94)

Ward et al. Blood

2008;111:5727-33.



(214.) Song SN, Tomosugi N, Kawabata H, Ishikawa T, Nishikawa T,

Yoshizaki K. This increased synthesis leads to a deficiency of

iron available for erythropoiesis, and is considered to be the mechanism

underlying reticuloendothelial (RE) iron sequestration, intestinal iron

absorption impairment, and low serum iron concentrations characteristic

of anemia of chronic disease.



(222.) Tjalsma H, Laarakkers CM, van Swelm RP, Theurl M, Theurl I,

Kemna EH, et al. Hepcidin was originally

identified as an antimicrobial peptide (1, 11). Fleming, [3]

and Dorine W. J Biol Chem 2001;276:7811-9.



(87.) Kobold U, Dulffer T, Dangl M, Escherich A, Kubbies M,

Roddiger R, Wright JA. Viral infection and iron

metabolism. The relatively high hepcidin

concentrations in some patients might be a consequence of the

transferrin saturation that remained increased despite phlebotomy. On the basis of theoretical

calculations, 11% of hepcidin was estimated to be freely circulating

(28). Additional data were reported that

suggested an atherogenic function of hepcidin in a study that revealed

that ex vivo hepcidin-25 treatment of differentiating monocytes from

patients with the metabolic syndrome induced monocyte chemoattractant protein-1 (192). Quantitation of

hepcidin from human and mouse serum using liquid chromatography tandem

mass spectrometry. Blood 2005;106: 3979-84.



(119.) Altamura S, D'Alessio F, Selle B, Muckenthaler MU. J Gastroenterol 2011; 46:648-56.



(190.) Barisani D, Pelucchi S, Mariani R, Galimberti S, Trombini P,

Fumagalli D, et al. In IDA patients, and also in patients with low

ferritin concentrations without anemia, secondary to blood losses or

insufficient dietary intake, hepcidin concentrations have been found to

be very low, often below the lower limit of detection of the employed

assays (13, 29).



(144.) Swinkels DW, Drenth JP. J Mol Med 2011.



[FIGURE 4 OMITTED]



(91.) Anderson DS, Heeney MM, Roth U, Menzel C, Fleming MD, Steen

H. Evidence for the latter explanation comes from the

finding of increases of only 1- to 6-fold of serum hepcidin

concentrations in patients with glomerular dysfunction (15, 16, 29, 31,

32) compared with the 20- to 30-fold increase of serum

[[beta].sub.2]-microglobulin. Prohepcidin: expression and cell specific

localisation in the liver and its regulation in hereditary

haemochromatosis, chronic renal insufficiency, and renal anaemia. Anal Biochem 2002;301:49-56.



(116.) del Giudice EM, Santoro. finding electric problems in a car can be challenging. It is very difficult to trace one of the hundreds of wires in a vehicle. You need a specialized electric automotive repair shop to find most electric problems in a vehicleN, Amato A, Brienza C, Calabro P,

Wiegerinck ET, et al. On the

other hand, increased hepcidin concentrations may lead to iron

deficiency and blunt the efficacy of iron fortification programs by

decreasing iron absorption (157). HIF-2alpha, but not HIF-1alpha, promotes iron absorption

in mice. Novel urine hepcidin assay by mass spectrometry. Acta Haematol

2009;122:226-9.



Joyce J.C. Hepcidin

agonists could be useful to prevent iron overload attributed to hepcidin

deficiency, such as HH, and especially [beta]-thalassemias and other

iron-loading anemias, and possibly some acquired forms of

non-hemochromatotic iron-overload diseases. J Mol Med 2010;88:477-86.



Recent observations suggest that the erythropoietic signal may

include 1 or more proteins released at sites of active erythropoiesis,

i.e., growth differentiation factor-15 (growth differentiation factor-15

(GDF15) and twisted gastrulation protein homolog-1 (TWSG1). 1) (3). It is likely that additional

erythropoietic factors downregulating hepcidin expression will be

identified.



(11.) Park CH, Valore EV, Waring AJ, Ganz T. Mass

spectrometry-based hepcidin measurements in serum and urine: analytical

aspects and clinical implications. Haematologica 2008;93:1473-9.



[1] Department of Laboratory Medicine, Laboratory of Genetic,

Endocrine and Metabolic Disorders, [2] Hepcidinanalysis.com, Radboud

University Nijmegen Medical Centre, Nijmegen, the Netherlands; [3] Saint

Louis University School of Medicine, St. Intracellular iron stores

communicate with hepcidin via BMPs, particularly BMP-6, in a paracrine or autocrine fashion. Severe iron

deficiency blunts the response of the iron regulatory gene Hamp and

proinflammatory cytokines to lipopolysaccharide. J Clin Invest

2007;117:1926-32.



Hepcidin in acquired forms of nonhemochromatotic iron overload.

Several nonhereditary liver diseases such as excessive alcohol

consumption, nonalcoholic fatty liver disease and nonalcoholic

steatohepatitis are associated with mild hepatic iron overload. Kroot, [1] Harold Tjalsma, [1,2] Robert E. Possibly hepcidin should be

regarded as a marker of response rather than resistance to ESA treatment

in these patients.



(7.) Melino S, Garlando L, Patamia M, Paci M, Petruzzelli R. Blood 2005;106:2196-9.



(177.) Bratescu LO, Barsan L, Munteanu D, Stancu S, Mircescu G. Am J Hematol

2011;86: 107-9.



(200.) Peeling P, Dawson B, Goodman C, Landers G, Wiegerinck ET,

Swinkels DW, Trinder D. Hepcidin as a predictor of response to epoetin therapy in

anemic cancer patients. Relationship of hepcidin with parasitemia and anemia among

patients with uncomplicated Plasmodium falciparum malaria in Ghana. Phlebotomies or erythropoietin injections allow mobilization

of iron stores in a mouse model mimicking intensive care anemia. However, the redox activity of iron can also

cause damage, primarily by the production of reactive oxygen radicals.

As such, iron concentrations must be tightly regulated both at the

cellular and the systemic level. Anemia of chronic disease. BMP6 treatment compensates for the

molecular defect and ameliorates hemochromatosis in Hfe knockout mice.

Gastroenterology 2010;139:1721-9.



For Hodgkin lymphoma we recently demonstrated that increased IL-6

production is associated with induction of hepcidin, which contributes

to the iron-restricted anemia of chronic disease often observed at

Hodgkin lymphoma diagnosis. Mass spectrometry analysis of hepcidin peptides in

experimental mouse models. Patients with most forms of

HH have the inability to appropriately upregulate hepcidin synthesis in

response to increased iron stores. Large and well-designed studies exploiting

harmonized assays are required to more firmly establish the position of

hepcidin in diagnostic medicine. Contribution of

STAT3 and SMAD4 pathways to the regulation of hepcidin by opposing

stimuli. Iron is

necessary for microbial growth, and reductions in plasma iron are

bacteriostatic. Nat Med 2007;13:1096-101.



Several physiologic and pathologic processes regulate the synthesis

of hepcidin (Fig. TLR4-dependent hepcidin expression by myeloid cells in response

to bacterial pathogens. Neither of these

factors, however, appears to be required to mediate the decrease in

hepcidin observed with EPO administration. Blood Cells Mol Dis 2008;40:339-46.



Montosi G, Chen S, et al. Blood 2011;117:997-1004.



(154.) de Mast Q, van Dongen-Lases EC, Swinkels DW, Nieman AE,

Roestenberg M, Druilhe P, et al. Consistent with this iron

loading, low hepcidin concentrations have been found in these patients,

along with other changes in the expression of iron-transport genes

[reviewed in (159)]. We

observed lower serum hepcidin concentrations in premenopausal women than

in postmenopausal women (median 4.1 nmol/L vs 8.5 nmol/L) (109).

Hepcidin concentrations in men were constant over age (median 7.8

nmol/L). Mutations in the gene encoding the cellular iron

exporter ferroportin can also cause HH. J Clin Endocrinol

Metab 2009;94:5102-7.



(219.) Xiao JJ, Krzyzanski W, Wang YM, Li H, Rose MJ, Ma M, et al.

Pharmacokinetics of anti-hepcidin monoclonal antibody Ab 12B9m and

hepcidin in cynomolgus monkeys. Hepcidin

in CKD also has been associated with iron-restricted erythropoiesis, as

reflected by the relation of hepcidin with lower hemoglobin and/or

reticulocyte counts (31, 169, 171). Blood

2009;114:493-4.



(64.) Wrighting DM, Andrews NC. Int J Obes 2009;33:1111-7.



(209.) Corradini E, Schmidt PJ, Meynard D, Garuti C,



HEPCIDIN AGONISTS



(164.) Thomas C, Kobold U, Balan S, Roeddiger R, Thomas L. However, these assays could be used

to measure hepcidin only in a semiquantitative manner.



(82.) Brookes MJ, Sharma NK, Tselepis C, Iqbal TH. Gardenghi et

al. As a result the outcomes of studies to assess the

diagnostic value of hepcidin largely have depended on the definition of

surrogate gold standards. The hepcidin-binding site on ferroportin is

evolutionarily conserved. Altogether, these limitations should be kept in mind

during the interpretation of urine hepcidin data and the decision to

develop dedicated point-of-care devices for urine hepcidin measurements.



(17.) Tessitore N, Girelli D, Campostrini N, Bedogna V, Pietro SG,

Castagna A, et al. In

general, these studies have reported increased hepcidin concentrations

in CKD patients compared with healthy controls (15, 16, 29, 31, 32, 88,

89, 96, 167-169). J Biol Chem 2011;286:4090-7.



(169.) Weiss G, Theurl I, Eder S, Koppelstaetter C, Kurz K,

Sonnweber T, et al. The utility of multivariate analysis

in the study of hepcidin. Hepcidin: from

discovery to differential diagnosis. International Conference on

Harmonisation of Technical Requirements for Registration of

Pharmaceuticals for Human Use: ICH harmonised tripartite guideline text

on validation of analytical procedures. Situations in which demand for

circulating iron is increased (particularly erythropoietic activity)

elicit a decrease in hepatocellular hepcidin synthesis. Resistance to hepcidin is conferred

by hemochromatosis-associated mutations of ferroportin. Hepcidin

expression and iron transport in alveolar macrophages. The excretion of the latter low molecular

weight protein is known to be almost completely governed by glomerular

filtration. Soluble

HJV, which also acts as an antagonist of BMP signaling, decreases

hepcidin baseline expression in mice, mobilizes RE-system (splenic) iron

stores, increases liver iron content and concurrently increases serum

concentrations (208). Erythropoietin administration in humans causes a marked and

prolonged reduction in circulating hepcidin. Whether HIFs directly bind to the

hepcidin promoter is currently controversial. Louis, MO.



(194.) Maes K, Nemeth E, Roodman GD, Huston A, Esteve F, Freytes C,

et al. The introduction of ESAs, such as EPO,

has allowed effective treatment of anemia in patients with CKD. Such pathogens are

common in malaria-endemic regions. In cases in which interference has occurred, assays must be

revalidated for this specific application before definite conclusions on

the efficacy of the therapy can be drawn. This latter observation suggests a role

for hepcidin in modulating acute inflammatory responses to bacterial

infection.



(178.) Ho J, Lucy M, Krokhin O, Hayglass K, Pascoe E, Darroch G, et

al. The prevalence of iron deficiency is

high among athletes. Haematologica

2010;95:505-8.



(93.) Bansal SS, Halket JM, Bomford A, Simpson RJ, Vasavda N, Thein

SL, Hider RC. Iron in arterial plaque: modifiable risk factor

for atherosclerosis. Am J Physiol Gastrointest Liver

Physiol 2006;291:G229-37.



(65.) Verga Falzacappa MV, Vujic SM, Kessler R, Stolte J, Hentze

MW, Muckenthaler MU. Erythropoietin mediates hepcidin expression in hepatocytes

through EPOR signaling and regulation of C/EBPalpha. Macrophage iron, hepcidin, and atherosclerotic

plaque stability. Simple and

sensitive quantification of bioactive peptides in biological matrices

using liquid chromatography/selected reaction monitoring mass

spectrometry coupled with trichloroacetic acid clean-up. It

appears that hepcidin values, like those of other hormones, should be

interpreted in the context of other indices of iron metabolism. High-sensitive radioimmunoassay for

human serum hepcidin. Haematologic data, iron parameters and molecular findings in

two new cases of iron-refractory iron deficiency anaemia. Defects in TfR2 and HFE lead to

decreased hepcidin concentrations via the extracellular signal-regulated

kinases: the mitogen-activated protein kinase (ERK/MAPK) pathwayand/or

the bone morphogenetic protein/mothers against decapentaplegic homolog (Drosophila) (BMP/SMAD) pathway (Fig. PLoS ONE 2011;6: e16762.



(126.) Piperno A, Girelli D, Nemeth E, Trombini P, Bozzini C,

Poggiali E, et al. (87)

Bansal et al. Evaluation of hepcidin isoforms in hemodialysis

patients by a proteomic approach based on SELDI-TOF MS. Further studies in which hepcidin response to iron

supplementation is used as a proxy for the gold standard of iron status

would be informative. Exercise as a mediator of hepcidin activity in

athletes. By

contrast, mutational defects that result in the loss of ferroportin

function cause cellular iron overload due to compromised iron export.

Such patients thus have some phenotypic characteristics which vary from

those of classical HH and have been reported to have high (urinary)

hepcidin concentrations when untreated (127).



We foresee the following potential applications of hepcidin

measurements in patients diagnosed with or suspected for HH: (a)

screening for the presence of HH in patients with increased ferritin

concentrations; (b) prioritization of genes to be investigated (very low

hepcidin concentrations point to juvenile HH forms); (c) prediction of

which C282Y-homozygous patients will be at risk for iron overload; and

(d) monitoring of phlebotomy treatment (123, 133).



(202.) Troadec MB, Laine F, Daniel V, Rochcongar P, Ropert M,

Cabillic F, et al. (43)



(a) UPLC, ultra-high pressure LC; MS/MS, tandem MS; Q,

quadrupole; HBD, hepcidin-binding domain.



Table 2. Recent advances in the understanding of iron overload in

sideroblastic myelodysplastic syndrome. Hepcidin concentrations have not been

consistently compared between races and there are few studies in

children (113-120). Mild increases in serum hepcidin and

interleukin-6 concentrations impair iron incorporation in haemoglobin during an experimental human malaria infection. Some studies showed that hepcidin is an

appropriate marker in the differentiation of ACD and IDA/ACD but not of

IDA and IDA/ACD (75), whereas others have reported that hepcidin allowed

distinguishing IDA from IDA/ACD but not ACD from IDA/ACD (13,164).

Results of another study have suggested that hepcidin concentrations can

serve as an appropriate marker to differentiate between all 3 groups

(165).



(131.) Wijngaarden P, Hoskam J, Koeken A, Boer JMA, Swinkels DW,

Ermens AAM, Cobbaert CM. Rheumatol Int 2010;30:917-23.



[FIGURE 1 OMITTED]



(196.) Zucker SD. Cell Metab 2008;8:146-56.



(74.) Darshan D, Frazer DM, Wilkins SJ, Anderson GJ. Am J Clin

Nutr 2009;90: 1280-7.



INTERACTION BETWEEN HEPCIDIN REGULATORY PATHWAYS



Several components can interfere with the BMP/ SMAD pathway.

Administration of dorsomorphin, a small inhibitor of BMP signaling,

prevented hepcidin induction by iron in mice (212). Blood 2007;110:4096-100.



(136.) Kearney SL, Nemeth E, Neufeld EJ, Thapa D, Ganz T, Weinstein

DA, Cunningham MJ. Anemia in Hodgkin's lymphoma: the role of

interleukin-6 and hepcidin. In these

so-called nonhemochromatotic iron overload diseases, hepcidin

concentrations are increased, but relatively low for the level of iron

overload, suggesting ineffective liver iron sensing. et al. Clin Chim Acta 2010;411:453-5.



(88.) Bansal SS, Abbate V, Bomford A, Halket JM, Macdougall IC,

Thein SL, Hider RC. Med Sci Sports Exerc

2009;41:1138-45.



Liver iron stores and circulating transferrin-bound iron

(Tf-[Fe.sub.2]) each provide distinct signals to influence hepatocyte hepcidin expression (44-46) and are considered separately (Fig. This difficulty is due

to the small and compact structure of hepcidin, which leaves scarce

antigenic epitopes (Fig. Nat Chem Biol 2008;4:33-41.



Human hepcidin is predominantly produced by hepatocytes as a 25

amino acid peptide (2789.4 Da) (1, 2), that is secreted into the

circulation. Hereditary haemochromatosis. Interleukin-6 induces hepcidin

expression through STAT3. Intravenous iron administration was found not to

influence hepcidin concentrations in hemodialysis (HD) patients that

were withheld from iron and ESA therapy for 2 weeks (169), whereas it

increased hepcidin in iron-naive CKD patients (31, 96). Studies in

patients with multiple myeloma suggest that hepcidin is upregulated in

these patients by both IL-6-dependent and IL-6-independent mechanisms

that may play a role in the anemia often observed in these patients. Moreover, the

response of the hepcidin to oral iron might prove to be a useful test to

evaluate iron absorption in iron deficiency.



(173.) Zaritsky J, Young B, Wang HJ, Westerman M, Olbina G, Nemeth

E, et al. Here, we review the major

advances from studies on the structure, kinetics, regulation, and

function of hepcidin and the current state-of-the-art in hepcidin

assays. Diagnosis and

management of hereditary haemochromatosis. Reduction of serum hepcidin by hemodialysis in pediatric and

adult patients. J Nutr 2001;131: 616S-33S.



(179.) Prowle JR, Westerman M, Bellomo R. Recombinant human hepcidin expressed in Escherichia

coli isolates as an iron containing protein. Bone morphogenetic protein signaling by hemojuvelin regulates

hepcidin expression. (96) (Bachem)

Grebenchtchikov et al. Pathophysiology of transfusional iron overload:

contrasting patterns in thalassemia major and sickle cell disease.

Hemoglobin 2009; 33(Suppl 1):S37-45.



Hepcidin-targeted therapies may improve treatment options for

patients suffering from iron disorders. Hepcidin might also contribute indirectly

to host defense by reducing plasma iron concentrations. The molecular basis of ferroportin-linked hemochromatosis.

Proc Natl Acad SciUSA 2005;102:8955-60.



To date, differences in hepcidin concentrations between the sexes

have not been consistently found (12, 29, 85, 96, 97, 107). Proc Natl Acad SciUSA2001;98: 8160-2.



(163.) Andrews NC. Blood

2011;117:e218-25.

. Exp Biol Med 2007; 232:1014-20.



Hepcidin Synthesis and Structure

(189.) Sullivan JL. Association of body iron stores with low

molecular weight iron and oxidant damage of human atherosclerotic

plaques. Repeated intravenous iron

infusions, on the other hand. Polymorphisms and mutations of human TMPRSS6 in iron deficiency

anemia. J Am Soc Nephrol 16,

49A 2005.



(161.) Fleming RE, Sly WS. Involvement of hepcidin in the anemia of multiple myeloma.

Clin Cancer Res 2008;14:3262-7.



(90.) Campostrini N, Castagna A, Zaninotto F, Bedogna V, Tessitore

N, Poli A, et al. (88)

LC-MS/MS Murphy et al. Mice lacking intestinal HIF2[alpha] have decreased expression of

both ferroportin and divalent metal transporter 1, the principal iron

importer at the apical surface of duodenal enterocytes, and thus fail to

induce iron absorption even upon lowering of hepcidin expression (63).

These data suggest that in hypoxic (or iron deficient) conditions,

expression of HIF2[alpha] promotes iron absorption from the intestine

via increased activity of divalent metal transporter 1 and ferroportin.

This result implies that HIF2[alpha]-induced changes in iron transport

can override the effects of the hepcidin-ferroportin regulatory axis.



(224.) Kemna EH, Tjalsma H, Willems HL, Swinkels DW. Iron

delocalisation in the pathogenesis of ma larial anaemia. Alcohol-dependent

downregulation of C/EBP[alpha], probably through radical oxygen species,

might be responsible for a decrease of hepcidin expression and

subsequent (mild) iron overload in heavy drinkers (69, 70).



Previously published online at DOI: 10.1373/clinchem.2009.140053



(40.) De Domenico I, Zhang TY, Koening CL, Branch RW, London N, Lo

E, et al. These

patients demonstrated an exaggerated increase in serum iron

concentrations upon oral iron loading (132). In a recent study among

elderly individuals ([greater than or equal to]65 years old), the

presence of anemia could not be attributed to increased hepcidin

concentrations, because urinary hepcidin concentrations were

significantly lower among participants with inflammation anemia and iron

deficiency compared with nonanemic controls. This dysregulation is caused by

defects in certain genes encoding for positive regulators of hepcidin

(HFE, TfR2, HJV), or, rarely, the hepcidin gene [hepcidin antimicrobial

peptide (HAMP)] itself [reviewed in (123, 124)]. (29)

Koliaraki et al. Reports on the effect

of a defective HFE/TfR2 pathway on hepcidin upregulation by inflammation

are not consistent. Urinary hepcidin in congenital chronic anemias.

Pediatr Blood Cancer 2007;48:57-63.



(115.) de Mast Q, Syafruddin D, Keijmel S, Olde RT, Deky O, Asih

PB, et al. J Leukoc Biol 2007;82:934-45.



Hepcidin Regulation



Although the number of studies on the role of hepcidin in the

anemia of cancer is limited, these studies have contributed to our

insights regarding the mechanisms involved. Haematologica 2010;95:1068-74.



(58.) Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I,

et al. Anemia of chronic heart failure has

been found to be associated with low hepcidin concentrations, making

(low-grade) inflammation less likely as the sole underlying mechanism

(184, 185).A study on acute cardiac ischemia showed a transient increase

in serum hepcidin-20 concentrations for the majority of patients

studied, whereas hepcidin-25 remained increased 7 days after the

incident. This situation implies

that until harmonization is achieved, reference intervals as well as

clinical decision limits for certain patient populations specific to the

assay methods should be used. Quantitation of hepcidin in human urine by liquid

chromatography-mass spectrometry. The presence of the iron exporter ferroportin at the plasma membrane

of macrophages is enhanced by iron loading and downregulated by

hepcidin. Serum hepcidin and macrophage iron correlate with

MCP-1 release and vascular damage in patients with metabolic syndrome

alterations. World J Gastroenterol 2007;13:4925-30.



Hepcidin in obesity-related diseases. Although the

smaller forms of hepcidin do not elicit a hypoferremic response, it is

currently unknown whether they retain other identified biological

functions of hepcidin-25 (e.g., in host defense or in metal binding) (4,

6, 11, 43).



(113.) Semrin G, Fishman DS, Bousvaros A, Zholudev A, Saunders AC,

Correia CE, et al. to propose that

exercise-induced increases in hemolysis and hepcidin expression may have

2 effects. Surprisingly, these results are not fully

consistent with those found for patients with CKD (see above), but

suggest that baseline hepcidin concentrations may play a potential role

as a predictive marker to identify patients who either are going to

respond adequately to EPO therapy or need to be excluded from anemia

correction with ESA owing to a low chance of responding.



(49.) Silvestri L, Pagani A, Nai A, De D, I, Kaplan J, Camaschella

C. For

instance, it is possible that "normal" concentrations of

hepcidin in iron deficiency anemia (IDA) are inappropriately high, and

perpetuate iron restriction.



(130.) Mayr R, Janecke AR, Schranz M, Griffiths WJ, Vogel W,

Pietrangelo A, Zoller H. Hepcidin serum levels and resistance to recombinant

human erythropoietin therapy in haemodialysis patients. Blood Cells Mol Dis 2002;29:

327-35.



(77.) Constante M, Jiang W, Wang D, Raymond VA, Bilodeau M, Santos

MM. 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. Am J Trop Med Hyg 2011;84: 148-51.



(217.) Kawabata H, Tomosugi N, Kanda J, Tanaka Y, Yoshizaki K,

Uchiyama T. Blood 2006;107:328-33.



(20.) Peyssonnaux C, Zinkernagel AS, Datta V, Lauth X, Johnson RS,

Nizet V. Interpretation of urinary hepcidin data is

difficult because urine concentrations may also depend on glomerular

filtration, tubular reabsorption, local production by tubular epithelial

cells (25), and production by interstitial inflammatory cells (108).

These issues make measurement of hepcidin somewhat disadvantageous in

urine compared to serum and imply that for reliable interpretation of

urine hepcidin concentrations as an alternative for serum

concentrations, information on renal hepcidin production and handling is

needed. Curr Opin Crit Care 2003;9:496-502.



(121.) Van Santen S, de Mast Q, Luty AJ, Wiegerinck ET, van der Ven

A, Swinkels DW. HH comprises several

genetic disorders of iron homeostasis characterized by body iron excess.

This results in body iron overload, which can progress to organ injury,

such as liver fibrosis and cirrhosis (122). Hepcidin-25 is a marker of the

response rather than resistance to exogenous erythropoietin in chronic

kidney disease/chronic heart failure patients. Nat Genet 2008;40: 569-71.



(9.) Schranz M, Bakry R, Creus M, Bonn G, Vogel W, Zoller H.

Activation and inactivation of the iron hormone hepcidin: biochemical

characterization of prohepcidin cleavage and sequential degradation to

N-terminally truncated hepcidin isoforms. Blood 2006;107:3727-32.



(52.) Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns

TD, et al. Cell Metab 2008;8:502-11.



* Address correspondence to this author at: Department of

Laboratory Medicine, Laboratory of Genetic, Endocrine and Metabolic

Diseases 830, Radboud University Nijmegen Medical Centre, P.O. Box 9101,

6500 HB Nijmegen, the Netherlands. (86)

Li et al. These

findings are in agreement with results of studies demonstrating that

high erythroid demand for iron blunts hepcidin induction by a

concomitant inflammatory response (50, 72-75). In vivo studies in mice have

demonstrated that only full-length 25 amino acid hepcidin induces

significant hypoferremia when injected intraperitoneally (18). J Biol Chem

2001;276:7806-10.



(142.) Cuijpers ML, Raymakers RA, Mackenzie MA, de Witte TJ,

Swinkels DW. As expected, patients with these mutations have a phenotype

similar to classical HH. In addition, Ganz et al.

(29) developed a competitive (c)ELISA for human serum hepcidin. Hemojuvelin

(HJV), a BMP coreceptor (47), is crucial for hepcidin expression because

various hepcidin regulatory pathways converge at this membrane-bound

protein. (92)



HBD assay De Domenico et al. J Pept Res 2005;66:65-71.



(45.) Ramos E, Kautz L, Rodriguez R, Hansen M, Gabayan V, Ginzburg

Y, et al. High-throughput matrix-assisted laser desorption ionization-time-of-flight mass spectrometry method for quantification of

hepcidin in human urine. Hepcidin in human iron disorders: therapeutic

implications. Loss of

matriptase-2 activity results in an inability to reduce hepcidin

synthesis during iron deficiency (49). Ferroportin disease: a systematic meta-analysis

of clinical and molecular findings. Quality control based on

isotopic distributions for high-throughput MALDI-TOF and MALDI-FTICR

serum peptide profiling. A dual-monoclonal sandwich ELISA specific for

hepcidin-25. Induction of erythropoiesis and iron utilization by the HIF

prolyl hydroxylase inhibitor FG-4592 [Abstract]. have been reported to (partially) increase

hemoglobin and mean cell volume (119, 120, 148-151). Blood 2008;111:2392-9.



(78.) van Deuren M, Kroot JJ, Swinkels DW. Haematologica

2010;96:500-6.



(25.) Kulaksiz H, Theilig F, Bachmann S, Gehrke SG, Rost D,

Janetzko A, et al. Another

potential drawback of urine hepcidin measurements is its sensitivity to

oxidation (30) and the relatively high concentration of the smaller

hepcidin isoforms, which cannot be distinguished from hepcidin-25 by

most immunoassays. The STARD initiative and the reporting of studies

of diagnostic accuracy. Serum hepcidin but not prohepcidin may be an effective marker for

anemia of inflammation (AI). These extracellular signaling molecules act on

hepatocellular BMP receptors to activate the intracellular SMAD signaling pathway and increase hepcidin transcription. Annu Rev Pathol 2009;4: 489-515.



(165.) Sasu BJ, Li H, Rose MJ, Arvedson TL, Doellgast G, Molineux

G. Hepcidin antagonists, on the

other hand, might be expected to benefit patients with diseases of

hepcidin excess manifested as iron-restricted anemia and systemic iron

deficiency, such as iron-refractory IDA, ACD (rheumatic diseases,

inflammatory bowel diseases, autoimmune diseases), CKD, multiple myeloma

and other cancers, obesity-related iron deficiency, and cardiovascular

disease.



STRENGTHS AND LIMITATIONS OF QUANTITATIVE PLASMA AND URINE HEPCIDIN

ASSAYS





Table 1. Moreover, well-designed large

clinical studies addressing safety and long-term efficacy are needed to

clarify the risks and benefits of hepcidin-targeted treatments.



(198.) Peeling P, Dawson B, Goodman C, Landers G, Trinder D.

Athletic induced iron deficiency: new insights into the role of

inflammation, cytokines and hormones. Although the physiological relevance of GDF15 and TWSG1 remains

to be defined, these proteins might have therapeutic potential. Biochem J 2010;431:363-71.



(33.) Sumboonnanonda A, Malasit P, Tanphaichitr VS, Ong-ajyooth S,

Sunthornchart S, Pattanakitsakul S. Nat Genet

2004;36:481-5.



(27.) Isoda M, Hanawa H, Watanabe R, Yoshida T, Toba K, Yoshida K,

et al. Rapid Commun Mass Spectrom 2010;24:1251-9.



(120.) Tchou I, Diepold M, Pilotto PA, Swinkels D, Neerman-Arbez M,

Beris P. This stress response can be controlled by the transcription

factor cAMP-response-element-binding-protein-H (67) or by the

stress-inducible transcription factors CCAAT-enhancer-binding protein

(C/EBP[alpha]) and C/EBP-homologous protein (68). Growth differentiation factor 15 in patients with

congenital dyserythropoietic anaemia (CDA) type II. Two to

tango: regulation of Mammalian iron metabolism. Obesity (Silver Spring)

2010;18:1449-56.



Recent studies have demonstrated hepcidin expression by cells other

than hepatocytes, although at much lower levels by comparison. Studies to assess the

role of hepcidin in ACD are complicated by the lack of a gold standard

methodology. These observations raise

the possibility that hepcidin may prove to be a novel determinant for

cardiovascular disease risk.



(59.) Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen

BK, Ganz T. Through an autocrine interaction with ferroportin, local hepcidin may protect the nearby

cells from iron deficiency, prevent extracellular oxidative stress,

affect inflammatory reponses, and/or deplete extracellular iron pools

that are available for extracellular pathogens (27, 40-42). J Clin Invest

2002;110:1037-44.



(54.) Pak M, Lopez MA, Gabayan V, Ganz T, Rivera S. Biochimie

2009;91:1223-8.



(168.) Zaritsky JJ, Young BY. Posttranslational processing of hepcidin

in human hepatocytes is mediated by the prohormone convertase furin.

Blood Cells Mol Dis 2008;40:132-8.



(152.) Oppenheimer SJ. It exerts its regulatory function

by counteracting the function of ferroportin, the major cellular iron

exporter in the membrane of macrophages, hepatocytes, and the

basolateral site of enterocytes. Blunted hepcidin response to oral iron challenge in

HFE-related hemochromatosis. Accordingly, patients have

increased innate hepcidin concentrations resulting in extremely low

transferrin saturation percentages and ferritin concentrations in the

lower range of the reference interval (118, 119, 120, 148-151).

Therefore, assessment of hepcidin concentrations can serve to exclude or

raise the suspicion for matriptase-2 deficiency in unexplained IDA and

thus help to avoid the unnecessary burden associated with multiple

diagnostic testing and delayed or ineffective treatment.



(21.) Bekri S, Gual P, Anty R, Luciani N, Dahman M, Ramesh B, et

al. Mutations in TMPRSS6 cause iron-refractory iron

deficiency anemia (IRIDA). In addition, the lower hepcidin concentrations observed

in response to hypoxia may be attributed to liver-specific stabilization

of hypoxia-inducible factor (HIF)-1 (60), with downstream effects on the

BMP/ SMAD signaling pathway (61, 62). Moreover, there is a need for harmonization of the various

assays to enable the establishment of world-wide reference intervals and

clinical decision limits. We furthermore summarize human studies of hepcidin in

physiological and pathophysiological states, and discuss the evidence of

the therapeutic potential of modulating hepcidin concentrations. These routes

comprise 4 highly interconnected regulatory pathways (Fig. This observation draws

further attention to the concern that lowering hepcidin concentrations

by phlebotomy in HH patients may exacerbate the underlying

pathophysiology, exacerbating the excess release of iron into the

circulation, with an ensuing vicious circle that leads to the need for

more frequent maintenance phlebotomies in HH patients (125, 126).



In a study among anemic cancer patients receiving ESA therapy,

those with relatively low hepcidin concentrations at baseline showed a

better response (197). Swinkels, NOXXON, Pharma AG,

Berlin, Germany; Pieris AG, Freising-Weihenstephan, Germany.



(134.) Kattamis A, Papassotiriou I, Palaiologou D, Apostolakou F,

Galani A, Ladis V, et al. J Clin Oncol 2010;28:2538-43.



The wide reference intervals may be attributed at least partly to

the increase of hepcidin concentrations in the course of the day (12,

29, 96, 97, 107). Regulation of iron homeostasis by the

hypoxiainducible transcription factors (HIFs). J Clin Invest 2010;120:4466-77.



(56.) Tanno T, Porayette P, Sripichai O, Noh SJ, By rnes C,

Bhupatiraju A, et al. Serum

hepcidin-25 may replace the ferritin index in the Thomas plot in

assessing iron status in auto repair anemic patients. To the best of our knowledge there are 2 reported

studies of hepcidin concentrations in pregnant women, both of which

showed that hepcidin concentrations in anemic pregnant women is lower

than in nonanemic pregnant women, but are not related to cord-blood or

newborn hepcidin or other fetal iron parameters (114, 121).



(149.) Melis MA, Cau M, Congiu R, Sole G, Barella S, Cao A, et al.

A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease

that suppresses hepcidin production, in familial iron deficiency anemia

refractory to oral iron. Second, increased hepcidin

activity postexercise may reduce the absorption of iron from the diet

(203). et al. Potential conflicts of interest:



(223.) Janssen MC, Swinkels DW. Br J Haematol 2010; 149:322-33.



(31.) Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns

TD, et al. Modelling of a metal-containing

hepcidin. J Hepatol

2008;49:123-33.



(166.) Swinkels DW, Wetzels JF. Whether binding to these carrier molecules influences the

functional properties of hepcidin is uncertain.



Small and large studies of healthy controls have revealed

considerable interindividual variation in hepcidin concentrations,

resulting in wide reference intervals (12, 29, 96, 97, 107, 109)