Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders.
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 monitoring 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 concentrations 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 measured with c-ELISA and WCX-TOF-MS differed; (b) hepcidin isoforms contributed to differences in hepcidin 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 concentrations <60 μg/L, and were suitable for distinguishing 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 concentrations, because of its low limit of detection, low cost, and high-throughput. Because of its specificity for bioactive 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.
Kroot
Joyce J C
JJ
Laboratory of Genetic, Endocrine and Metabolic Diseases, Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Laarakkers
Coby M M
CM
Geurts-Moespot
Anneke J
AJ
Grebenchtchikov
Nicolaï
N
Pickkers
Peter
P
van Ede
Annelies E
AE
Peters
Hilde P E
HP
van Dongen-Lases
Edmée
E
Wetzels
Jack F M
JF
Sweep
Fred C G J
FC
Tjalsma
Harold
H
Swinkels
Dorine W
DW
D000740:Anemia / Q000097:blood / Q000175:diagnosis* / Q000209:etiology; D018798:Anemia, Iron-Deficiency / Q000097:blood / Q000175:diagnosis / Q000209:etiology; D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000150:complications; D002908:Chronic Disease; D003937:Diagnosis, Differential; D004797:Enzyme-Linked Immunosorbent Assay; D064451:Hepcidins; D006801:Humans; D019189:Iron Metabolism Disorders / Q000097:blood / Q000175:diagnosis*; D013058:Mass Spectrometry; D020033:Protein Isoforms / Q000097:blood
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D020033:Protein Isoforms