Hepcidin Today Rachel van Swelm 10 05 2018 ISLH www.radboud ironcenter.com; www.hepcidinanalysis.com Discovery Hepcidin: discovery June 2000: Man: Plasma ultrafiltrate Liver Expressed Antimicrobial Peptide September 2000: Man: from urine hepcidin April 2001: Mice: Link to iron metabolism March 2001 Mice: increased by inflammation and iron overload 1
Structural characteristics Structural characteristics Properties: Peptide hormone Defensine like Production foremost hepatocytes 4 isoforms: 25-, 24-, 22- en 20-aa 4 disulphide bridges Highly conserved between species Charged, acidic Hepcidin 25 (m/z 2789.4) Charged, basic S-S bonds E. Kem na Park et al. J Biol Chem 2001; Hunter et al. J Biol Chem 2002; Melino et al. J Peptide Res 2006; Jordan et al. J Biol Chem 2009 Regulation and mode of action 2
Major body iron flows ~2500 mg Kroot et al. Clin Chem 2011 Systemic iron homeostasis: iron uptake and recycling ~2500 mg Kroot et al. Clin Chem 2011 Regulatory pathways of hepcidin Erythropoiesis po 2 stfr HIF 1α EPO TfR1 Kidney Bone marrow GDF15 TWSG1? Cell membrane Liver cell Iron status Circulating Iron Iron stores Fe 2 Transferrin Inflammation Matriptase 2 IL 6 HJV BMP 6 IL 6R HFE β 2M TfR2 TfR1 BMPR JAK SMAD STAT? Hepcidin Pro Hepcidin Nucleus HAMP 19 q 13 Promoter Kroot et al., Clin Chem 2011; Fleming et al. NEJM 2011; Hentze et al. Cell 2010 3
Hepcidin and ferroportin: mode of action Enterocyte Hepatocyte Diferric transferrin Hepcidin Macrophage Swinkels et al. Clin Chem 2006; Nemeth et al. Science 2004 Summary Hepcidin: discovery, structure, mode of action and regulation Hepcidin: was discovered in man and mice around 2000, as the long sought after regulator of iron metabolism is a 25 amino acid protein, 4 disulfide bridges, highly conserved peptide hormone is mainly produced by the liver is regulated by 3 regulatory pathways: erythropoietic activity (hypoxia), inflammation, body iron stores, circulating iron interacts with cellular iron exporter ferroportin in mainly enterocyte and macrophages and thereby decreases iron efflux and serum iron levels Girelli et al. Blood 2016 Measurements in human plasma 4
Development and optimization of hepcidin assays 2005 2013 Principles of hepcidin assays Hepcidin isoforms by mass spectrometry Weak Cation exchange MALDI Time of Flight Laarakkers PLoS ONE 2013 5
ELISA versus mass spectrometry Characteristic Costs Lower limit of detection Reproducibility Specificity Remarks ELISA costs less Depending on antibody and MS methodology MS generally better ELISA measures total hepcidin; MS measures hepcidin 25 Kroot et al. Clin Chem 2010; Galesloot et al. Blood 2011; Kroot et al. Clin Chem 2011; Laarakkers et al. PLoS ONE 2013; www.hepcidinanalysis.com Pre analysis and biological variation Pre analytical variation: recommendations 1. Hepcidin sticks to laboratory plastics: use small surface/volume ratio use serum/plasma and not urine 2. Hepcidin desintegrates at room temperature do not leave samples with hepcidin > 6 8 hrs at room temperature 3. Hepcidin in (internal) standard may desintegrate or stick to lab plastic take care in handling of hepcidin standards 6
Biological variation: circadian rhythm Serum Hepcidin Urine Hepcidin Serum and urine increase during the day, whereas iron decreases Kemna, Clin Chem 2007; Ganz, Blood 2008; Ashby, Kidney Int. 2008; Kroot, Anal Biochem 2009; Troutt, Clin Chem 2012 Harmonization and standardization World wide variety in hepcidin assays More info in Girelli, Nemeth and Swinkels, Blood 2016, supplemental Table S1. 7
Harmonization and standardization Primary reference material Harmonization True value Standardization Equivalent results Commutable secondary reference material Study 1 and 2 : Kroot J et al. Haematologica 2009; Am J Hemat 2012; Study 3: Van der Vorm, et al. Clin Chem 2016 Study 4: Diepeveen L et al. Establishment of a traceability chain to SI units for serum hepcidin allows standardization of serum hepcidin assays, manuscript in preparation. Clinical application of hepcidin assays Reference ranges for serum hepcidin (nm) Men (N=1,066) Women (N=882) 95% reference 95% reference range range P2.5 P97.5 P2.5 P97.5 Age, years N Median N Median 18-24 10 9.1 2.3 17.8 21 2.6 0.7 10.5 25-29 16 8.4 0.5 24.2 28 3.1 0.6 11.0 30-34 18 7.4 0.8 25.0 24 3.9 0.2 21.0 35-39 22 6.4 0.7 19.4 36 3.3 0.5 16.0 40-44 19 10.2 1.6 17.8 65 4.8 0.3 24.2 45-49 76 8.2 1.3 21.0 110 3.5 0.3 14.6 50-54 106 7.0 0.3 22.0 140 5.4 0.4 22.8 55-59 173 7.7 0.4 24.8 129 8.5 0.8 21.7 60-64 179 7.9 0.3 22.7 137 8.2 1.2 27.3 65-69 186 9.0 0.5 22.2 95 8.4 1.4 22.6 Median: 7.8 nm Median: 4.1 nm Median: 8.5 nm 70-74 133 8.4 1.0 26.9 62 8.7 1.0 37.8 75-79 99 6.8 0.8 25.5 16 9.2 2.1 29.0 80-84 22 6.8 3.5 20.1 10 11.9 1.6 19.2 85 7 11.3 3.4 20.5 9 6.7 1.2 24.5 All 1,066 7.8 0.6 23.3 882 6.5 0.5 23.2 Galesloot et al. Blood 2011; hepcidinanalysis.com 8
Iron deficiency anemia: low hepcidin Erythrocytes 2500 mg -- Camaschella C. NEJM 2015; Zimmermann MB et al. Lancet 2007 Mechanism of functional iron deficiency Fe Inflammation Fe Erythrocytes 2500 mg Fe Liver Anemia excess hepcidin iron withholding in macrophages anemia Anemia of inflammation/ iron deficiency Patients with rheumatoid arthritis IDA, iron deficiency anemia* ACD, anemia of chronic disease* *Using definition Weiss et al. NEJM 2005 van Santen et al, Arthritis & Rheumatism 2011; Girelli, Nemeth, Swinkels, Blood 2016 9
A B C 9 5 2018 IRIDA: elevated hepcidin functional iron deficiency Fe Fe TMPRSS6 Fe Anemia Anemia, low circulating iron (TSAT) and low normal RES stores (ferritin) Finberg, 2008; Du, 2008; Donker, 2014 TSAT/hepcidin as a diagnostic test in the diagnosis of IRIDA TSAT/hepcidin ratio (%/nm) 100 10 1 p=0.013 p=0.006 A. Bi allelic affected patients B. Mono allelic affected patients C. Relatives without an IRIDA phenotype 0.1 Boxes, median and interquartile ranges; whiskers, min to max TSAT, transferrin saturation Donker A. et al. Dutch IRIDA case series; Am. J. Hematol 2016 Hemochromatosis: innate low hepcidin -- Nemeth et al. Blood 2003 and 2004; Papanikolaou et al., Nat Genet. 2004; Kemna et al. Blood 2005; Kemna et al. Clin Chem 2007; van Dijk et al. British J. Haematol 2008; Girelli et al. Haematologica 2011; Tanno, Blood 2009; Tanno, Nat Med 2007; Kautz, Nat Genet 2014; Pak, Blood 2006; Origa, Haematologica 2015 10
Hereditary hemochromatosis Serum hepcidin (nm) Serum hepcidin/ferritin (pm/mug) van Dijk et al. British J Haemat 2008 Increased or ineffective erythropoiesis phlebotomy/blood donation EPO treatment ineffective erythropoiesis Erythroferrone GDF15 TWSG1 BMP/ SMAD -- Tanno, Blood 2009; Tanno, Nat Med 2007; Kautz, Nat Genet 2014; Pak, Blood 2006; Origa, Haematologica 2015 Development of hepcidin agonist and antagonists Erythropoiesis po 2 stfr HIF 1α EPO TfR1 Kidney Bone marrow GDF15 TWSG1? Cell membrane Liver cell Iron status Circulating Iron Iron stores Fe 2 Transferrin Inflammation Matriptase 2 IL 6 HJV BMP 6 IL 6R HFE β 2M TfR2 TfR1 BMPR JAK SMAD STAT? Hepcidin Hepcidin Pro Hepcidin Nucleus HAMP 19 q 13 Promoter Crielaard BJ et al. Nature Review 2017 Table 3; Fung E et al. Haematologica 2013 11
Many clinical conditions influence hepcidin levels: GFR CKD Dialysis RBC transfusions i.v. /oral iron replete iron Administration stores _ hepcidin _ Genetic factors (TMPRSS6 variants) Infectious/Inflammat ory Diseases Estrogens testosterone Ineffective/expanded erythropoiesis Anemia/hypoxia Erythropoietic Chronic liver disease Alcohol abuse (particularly Stimulating agents with synthetic IDA) Function Chronic HCV Infection Genetic factors (variants in HH genes) Clinical applications of hepcidin assay: most promising 1. Evaluation of suspected Iron Refractory Iron Deficiency Anemia (TSAT/hepcidin ratio) 2. Evaluation of iron overload disorders (hepcidin/ferritin ratio) 3. Diagnosis of concomitant iron deficiency in patients with anemia of chronic disease (low/intermediate/high hepcidin) 4. Guide for iron therapy 5. Monitoring of treatments targeting the hepcidin/ferroportin axis Before ordering the assay 1. Ensure local availability of a validated assay 2. Ensure control of pre analytical conditions (including diurnal rhythm) 3. Refer to age and sex specific ranges 4. Interpret hepcidin value into a minimum laboratory context (CBC, ferritin, transferrin saturation, CRP, serum creatinine, liver function tests). 5. Be aware of many potential confounders/comorbidities in the individual patient 12
Achievement of the hepcidin community since 2000 Discovery of iron regulatory peptide Fundamental studies on structure, regulation and mode of action improved understanding of (patho)physiology of iron metabolism Development and optimization of human assays Assessment of pre analytical factors, biological variation, reference values diagnostic performance Secondary reference material is produced and made available Ganz, Blood 2011; Girelli, Nemeth, Swinkels, Blood 2016 Research agenda Preparation of standardization assignment of value to secondary reference material by primary reference material World wide standardization; define clinical decision limits World wide external proficiency program Large studies on diagnostic performance Tissue specific functions of hepcidin Acknowledgements Prof. Dorine W. Swinkels Radboudumc, Nijmegen, The Netherlands Department of Laboratory Medicine Hepcidinanalysis.com Radboud Center for Iron Disorders 13