title: Mass spectrometry measurement of plasma hepcidin for the prediction of iron overload.
abstract: BACKGROUND
  Hepcidin has emerged as the primary regulator of iron homeostasis. Previous studies on assessing urinary hepcidin are limited. We developed a method for quantifying hepcidin-25 (Hep-25) in plasma using surface-enhanced laser-desorption-ionization time-of-flight mass spectrometry (SELDI-TOF/MS) and a 25-AA peptide as reference standard. The aims of the study were 1) to assess the performance of this method in different conditions of iron metabolism disorders; 2) to assess the diagnostic validity of non-invasive serum biomarkers in the identification of iron overload.
  
  
  METHODS
  Validation of the method was performed in 10 patients with type I hemochromatosis (HE) and in 177 subjects previously enrolled in a general population epidemiological study. Among the latter group, 17 had non-alcoholic fatty liver disease, 10 had chronic hepatitis C, and 150 subjects had normal ultrasound, normal liver function tests (LFTs), an alcohol intake < 20 g ethanol/day and were negative for the C282Y mutation. The following biomarkers were assayed in each case: plasma Hep-25, C282Y and H63D mutations of the HFE gene; serum iron, ferritin (SF), transferrin saturation, transaminases, γ-glutamyltransferase (GGT), glucose, insulin, total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglycerides.
  
  
  RESULTS
  Plasma Hep-25 concentrations were higher in HCV+ patients (26.3 ± 7.2 nmol/L) than in controls, and correlated positively with SF (p < 0.001). H63D heterozygous subjects revealed a pattern of iron overload that was significantly higher than H63D wild type subjects. Analyzing the data with the Biomarker Pattern 5.0.2. software to identify the most significant biomarkers for discriminating between HE cases and controls allowed us to produce an algorithm with four terminal nodes, which included glucose > 4.8 mmol/L and Hep-25/SF ratio ≤ 6.6 as the main splitters. These variables enabled the correct diagnosis of HE with 100% sensitivity, 93% specificity and an area under the receiver operating characteristic (ROC) curve of 0.993.
  
  
  CONCLUSIONS
  Our plasma Hep-25 mass spectrometry method yields measurements that reflect pathological and genetic influences; simple non-invasive biomarkers (Hep-25/SF ratio and glucose) can predict the presence of HE.
authors.affiliation: Department of Surgical and Gastroenterological Sciences, University of Padova, Padova, Italy. annarosa.floreani@unipd.it
authors.forename: Annarosa
authors.initials: A
authors.lastname: Floreani
authors.forename: Filippo
authors.initials: F
authors.lastname: Navaglia
authors.forename: Erik Rosa
authors.initials: ER
authors.lastname: Rizzotto
authors.forename: Daniela
authors.initials: D
authors.lastname: Basso
authors.forename: Maria
authors.initials: M
authors.lastname: Chiaramonte
authors.forename: Andrea
authors.initials: A
authors.lastname: Padoan
authors.forename: Ioannis
authors.initials: I
authors.lastname: Petridis
authors.forename: Nora
authors.initials: N
authors.lastname: Cazzagon
authors.forename: Roberto
authors.initials: R
authors.lastname: Testa
authors.forename: Maurizio
authors.initials: M
authors.lastname: Marra
authors.forename: Mario
authors.initials: M
authors.lastname: Plebani
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis / Q000379:methods*; D002648:Child; D064451:Hepcidins; D006801:Humans; D019190:Iron Overload / Q000097:blood* / Q000175:diagnosis*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D023181:Antimicrobial Cationic Peptides; D015415:Biomarkers; C578702:HAMP protein, human; D064451:Hepcidins