title: Mass spectrometry analysis of hepcidin peptides in experimental mouse models.
abstract: The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1) and its paralogue Hepcidin-2 (Hep-2) at the peptide level. To this purpose, Fourier transform ion cyclotron resonance (FTICR) and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF) MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i) 3 mouse strains (C57Bl/6; DBA/2 and BABL/c) upon stimulation with intravenous iron and LPS, ii) homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X) mutated mice and double affected mice, and iii) mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.
authors.affiliation: Laboratory of Genetic, Endocrine and Metabolic Diseases, Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Harold
authors.initials: H
authors.lastname: Tjalsma
authors.forename: Coby M M
authors.initials: CM
authors.lastname: Laarakkers
authors.forename: Rachel P L
authors.initials: RP
authors.lastname: van Swelm
authors.forename: Milan
authors.initials: M
authors.lastname: Theurl
authors.forename: Igor
authors.initials: I
authors.lastname: Theurl
authors.forename: Erwin H
authors.initials: EH
authors.lastname: Kemna
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Bas E
authors.initials: BE
authors.lastname: Dutilh
authors.forename: Frans G M
authors.initials: FG
authors.lastname: Russel
authors.forename: Günter
authors.initials: G
authors.lastname: Weiss
authors.forename: Rosalinde
authors.initials: R
authors.lastname: Masereeuw
authors.forename: Robert E
authors.initials: RE
authors.lastname: Fleming
authors.forename: Dorine W
authors.initials: DW
authors.lastname: Swinkels
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000652:urine; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects; D006432:Hemochromatosis / Q000097:blood / Q000235:genetics; D064451:Hepcidins; D015850:Interleukin-6 / Q000097:blood; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000187:drug effects / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; C579599:Hamp protein, mouse; C474320:Hamp2 protein, mouse; D064451:Hepcidins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D007501:Iron