title: Design and validation of a high-throughput matrix-assisted laser desorption ionization time-of-flight mass spectrometry method for quantification of hepcidin in human plasma.
abstract: Disorders of iron metabolism affect over a billion people worldwide. The circulating peptide hormone hepcidin, the central regulator of iron distribution in mammals, holds great diagnostic potential for an array of iron-associated disorders, including iron loading (β-thalassemia), iron overload (hereditary hemochromatosis), and iron deficiency diseases. We describe a novel high-throughput matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry assay for quantification of hepcidin in human plasma. This assay involves enrichment using a functionalized MALDI chip, a novel solvent-detergent precipitation buffer, and quantification using a stable isotope labeled internal standard. The linear range of hepcidin in plasma was 1-120 nM, with a low limit of quantification (LOQ) (1 nM), high accuracy (<15% relative error (RE)), and high precision (intraday average 5.52-18.48% coefficient of variation (CV) and interday 9.32-14.83% CV). The assay showed strong correlation with an established hepcidin immunoassay (Spearman; R(2) = 0.839 n = 93 ethylenediaminetetraacetic acid (EDTA) plasma). A collection of normal healthy pediatric samples (range 3.8-32.5 ng/mL; mean 12.9 ng/mL; n = 119) showed significant differences from an adult collection (range 1.8-48.7 ng/mL; mean 16.1 ng/mL; n = 95; P = 0.0096). We discuss these preliminary reference ranges and correlations with additional parameters in light of the utility and limitations of hepcidin measurements as a stand-alone diagnostic and as a tool for therapeutic intervention.
authors.affiliation: Departments of Pathology, Children's Hospital Boston, Boston, Massachusetts 02115, United States.
authors.forename: Damon S
authors.initials: DS
authors.lastname: Anderson
authors.forename: Marc
authors.initials: M
authors.lastname: Kirchner
authors.forename: Mark
authors.initials: M
authors.lastname: Kellogg
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Kalish
authors.forename: Jee-Yeong
authors.initials: JY
authors.lastname: Jeong
authors.forename: Gary
authors.initials: G
authors.lastname: Vanasse
authors.forename: Nancy
authors.initials: N
authors.lastname: Berliner
authors.forename: Mark D
authors.initials: MD
authors.lastname: Fleming
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
mesh_terms: D000328:Adult; D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D002648:Child; D005260:Female; D006432:Hemochromatosis / Q000175:diagnosis; D064451:Hepcidins; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay; D008297:Male; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins