Prediction of Iron Overload with a New Mass Spectrometry Method for Detection of Hepcidin in Plasma
Filippo Navaglia*1, Erik Rosa Rizzotto2, Daniela Basso1, Andrea Padoan1, Roberta Venturini1, Maria Chiaramonte3, Ioannis Petridis3, Roberto Testa4, Maurizio Marra4, Annarosa Floreani2, Mario Plebani1,5
1Laboratory Medicine, University-Hospital of Padua, Padua, Italy; 2Surgical and Gastroenterological Sciences, University of Padua, Padua, Italy; 3Medicine and Public Health, University of L’Aquila, L’Aquila, Italy; 4Gerontology Research, Diabetology Unit, INRCA, Ancona, Italy; 5Medical Diagnostic Sciences and Special Therapies, University of Padua, Padua, Italy
Hepcidin (Hep) has emerged as the primary regulator of iron homeostasis. Aims: 1. to assess the performance of plasma Hep measured by a SELDI-TOF-MS method in different conditions of iron metabolism disorders with respect to healthy controls (HC); 2. to identify, among a large series of metabolic, liver and iron metabolism indices, including Hep, those of potential diagnostic utility. Patients: 10 type I hemochromatosis (HH); 17 NAFDL; 10 HCV and 155 HC (normal ultrasound, normal LFTs, alcohol assumption <20 g ethanol/day). Plasma Hep was measured by SELDI-TOF-MS (reference standard = known concentrations of the synthetic 25-aa peptide). The following were included: glucose, insulin, C-peptide, HbA1c, cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, ferritin, transferrin, Fe++, TIBC, ALT, GGT, Hb, Hct, MCV, Ca++, uric acid, urea nitrogen, total proteins. Hep was higher in HCV (26.3±7.2 nmol/L, mean±SE) than in HC (12.3±1.0) (F=3.2, p<0.05). Fe++ and TIBC were higher in both HH and HCV with respect to HC (F=7.9, p<0.001 and F=23.7, p<0.001), while ferritin and transferrin were associated with HH only (F=7.0, p<0.001 and F=7.8, p<0.001). Glucose, C-peptide and insulin were higher in HH and NAFDL with respect to HC (F=21.1, p<0.001, F=18.1, p<0.001 and F=21.9, p<0.001). Hep was correlated with ferritin (r=0.451, p<0.001) and the index Hep/ferritin (H/F) was reduced in HH patients only (F=2.18, p=0.07). H63D heterozygotes subjects revealed a pattern of iron overload [higher Fe++ (t=2.7, p<0.05), TIBC (t=3.5, p<0.01), and lower H/F (t=2.5, p<0.05)] compared to H63D wild type subjects. To identify the indices which better allow to classify HH and NAFDL, the Biomarker Pattern software was employed. HH vs HC without H63D mutated allele: the best algorithm included as main splitters H/F, glucose and Fe++, which allowed to correctly classify 100% HH and 98.6% HC (AUC =0.993). NAFDL vs controls: the best algorithm included as main splitters insulin and glucose, which allowed to correctly classify 94% NAFDL and 89% HC (AUC =0.951). In conclusion: the new plasma Hep mass spectrometry method yields accurate measurements which reflect pathologic and genetic influences; simple non invasive markers (H/S, glucose and iron insulin and glucose) can suggest the presence of HH or NAFDL.
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