Measurement of Hepcidin-20, -22, -24, and-25 in Human Serum by Liquid Chromatography-High Resolution-Mass Spectrometry and its Clinical application

摘要

Hepcidin-25 is regarded as the master regulator of iron homeostasis. Three N-truncated isoforms of hepcidin-25 have been identified in human serum; hepcidin-20, -22, and -24, although information is scant as to the serum concentrations of these isoforms. A liquid chromatography-high resolution-mass spectrometry (LC-HR-MS) assay was developed for the simultaneous quantitation of hepcidin isoforms in human serum. Serum (200 μL) was mixed with aqueous formic acid (600 μL), and the supernatant loaded onto a 96-well- SPE-plate. Eluted sample (70 μL) was diluted with deionised water (60 μL) and analysed using LC–HR–MS. Samples previously analysed by a published LC-MS/MS assay were analysed for method comparison. All hepcidin isoforms were quantified in samples from healthy volunteers as controls, and patients with hereditary haemochromatosis (HH), non-alcoholic fatty liver disease (NAFLD), iron deficient anaemia (IDA), anaemia of chronic disease (ACD), and sickle cell anaemia (SCA). Samples were also analysed from individuals with chronic kidney disease (CKD) not requiring haemodialysis, and those pre- and post-haemodialysis. Intra-/inter-assay accuracy and precision were acceptable, calibration was linear (R2 0.90, all analytes), and the LLoQ was 1 μg/L (all analytes). There was a good correlation for hepcidin-25 to a published LC-MS/MS assay (y = 0.85x -3.2, R2 = 0.96). Median (range) hepcidin-25 concentrations in controls, and individuals with IDA, SCA, HH, ACD and sepsis were: 8 (1–31), 1 (1–2), 1 (1–10), 2 (1–15), 60 (10–213), and 92 (11–216) μg/L, respectively. Hepcidin-20, -22, or -24 were not detected in any control sample, but were detected in 30–100 % of all samples at 10–20 % of the hepcidin-25 concentration. Following haemodialysis, all hepcidin isoforms declined by some 35–50 %. Hepcidin-25 was most strongly correlated to hepcidin-24, and less so to hepcidin-22 and -20, in all disease states. The developed method was applicable for clinical use. However, further controlled studies are required to fully evaluate the role of hepcidin-20, -22, and -24 measurement in a clinical setting.