Immunochemical and Mass-Spectrometry–Based Serum Hepcidin Assays for Iron Metabolism Disorders Joyce J.C. Kroot, 1 Coby M.M. Laarakkers, 1 Anneke J. Geurts-Moespot, 1 Nicolaı¨Grebenchtchikov, 1 Peter Pickkers, 2 Annelies E. van Ede, 3 Hilde P.E. Peters, 4 Edme ´ e van Dongen-Lases, 1 Jack F.M. Wetzels, 4 Fred C.G.J. Sweep, 1 Harold Tjalsma, 1,5 and Dorine W. Swinkels 1,5* BACKGROUND: Hepcidin is an iron-regulatory peptide hormone that consists of 3 isoforms: bioactive hepcidin-25, and inactive hepcidin-22 and hepcidin- 20. Hepcidin is instrumental in the diagnosis and mon- itoring of iron metabolism disorders, but reliable methods for its quantification in serum are sparse, as is knowledge of their relative analytical strengths and clinical utility. METHODS: We developed a competitive (c)-ELISA and an immunocapture TOF mass-spectrometry (IC-TOF- MS) assay. Exploiting these 2 methods and our previously described weak cation exchange (WCX)- TOF-MS assay, we measured serum hepcidin concen- trations in 186 patients with various disorders of iron metabolism and in 23 healthy controls. RESULTS: We found that (a) the relative differences in median hepcidin concentrations in various diseases to be similar, although the absolute concentrations mea- sured with c-ELISA and WCX-TOF-MS differed; (b) hepcidin isoforms contributed to differences in hepci- din concentrations between methods, which were most prominent in patients with chronic kidney disease; and (c) hepcidin concentrations measured by both the c-ELISA and IC-TOF-MS correlated with ferritin con- centrations 60 g/L, and were suitable for distin- guishing between iron deficiency anemia (IDA) and the combination of IDA and anemia of chronic disease. CONCLUSIONS: c-ELISA is the method of choice for the large-scale quantification of serum hepcidin concen- trations, because of its low limit of detection, low cost, and high-throughput. Because of its specificity for bio- active hepcidin-25, WCX-TOF-MS can be regarded as a valuable special-purpose assay for disorders with variable concentrations of hepcidin isoforms, such as chronic kidney disease. © 2010 American Association for Clinical Chemistry Hepcidin is a hepatocyte-produced peptide hormone that regulates systemic iron homeostasis (1, 2 ). The mature bioactive form of hepcidin is a 25 amino acid peptide. Other isoforms in human blood and urine are the N-terminal truncated hepcidin-20 and -22 pep- tides, which are without apparent biological function (3). By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake, and mobilization from stores to meet the body iron need (1, 2 ). Hepcidin concentrations are decreased in con- ditions that demand increased serum iron concentra- tions (i.e., increased erythropoietic activity and iron deficiency), whereas concentrations are increased in infection and inflammation (4, 5 ). Since the discovery of hepcidin and its crucial role in iron homeostasis, there has been substantial interest in developing reliable assays to measure hepcidin con- centrations in body fluids. Accurate assessment of hep- cidin concentrations in serum would improve our un- derstanding of iron metabolism disorders and allow hepcidin to become a useful tool in the differential di- agnosis and clinical management of these diseases. Few investigative tools have been available for measuring hepcidin in biological fluids (6–9). We and others have reported related serum hepcidin quantifi- cation methods (10 –18 ). Assays based on mass spec- trometry (MS) 6 require relatively expensive equip- ment, but these methods are advantageous because they can be used to distinguish between hepcidin-25, -22, and -20. Immunoassays, on the other hand, are 1 Laboratory of Genetic, Endocrine and Metabolic Diseases, Department of Lab- oratory Medicine, 2 Department of Intensive Care Medicine, 3 Department of Rheumatology, and 4 Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 5 Hepcidinanalysis.com; Geert Grooteplein, Nijmegen, the Netherlands. * Address correspondence to this author at: Department of Laboratory Medicine, Laboratory of Genetic Endocrine and Metabolic Diseases 441, Radboud Uni- versity Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands. Fax 31-(0)24-3541743; e-mail d.swinkels@labgk.umcn.nl. Received May 11, 2010; accepted July 26, 2010. Previously published online at DOI: 10.1373/clinchem.2010.149187 6 Nonstandard abbreviations: MS, mass spectrometry; c-ELISA competitive ELISA, IC, immunocapture; WCX, weak cation exchange; IDA, iron deficiency anemia; ACD, anemia of chronic diseases; MM, multiple myeloma; HH, hereditary hemochromatosis; CKD, chronic kidney disease; RA, rheumatoid arthritis; LLOD, lower limit of detection; pI, isoelectric point. Clinical Chemistry 56:10 1570–1579 (2010) Proteomics and Protein Markers 1570