LC-MS/MS method for hepcidin-25 measurement in human and mouse serum: clinical and research implications in iron disorders
-
Thibaud Lefebvre
and
Zoubida Karim
Abstract
Background: The peptide hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. This 25-amino acid hormone is produced and secreted predominantly by hepatocytes. Hepcidin has been suggested as a promising diagnostic marker for iron-related disorders. However, its accurate quantification for clinical use remains so far challenging. In this report we describe a highly specific and quantitative serum hepcidin method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Material: The analytical validation included the determination of the limit of detection, of quantification, repeatability, reproducibility and linearity. This assay was developed for human and mouse hepcidin. The human assay was performed on serum patients with unexplained microcytic anemia. We applied our LC-MS/MS method for quantifying hepcidin-1 in mouse in various conditions: inflammation, hemolytic anemia, Hamp-1, Hjv and Hfe KO mice.
Results: We show that the LC-MS/MS is suitable for accurate determination of hepcidin-25 in clinical samples, thereby representing a useful tool for the clinical diagnosis and follow-up of iron-related diseases. In mouse, a strong correlation between hepatic Hamp-1 mRNA expression and serum hepcidin-1 levels was found (r=0.88; p=0.0002) and the expected variations in mouse models of iron disorders were observed.
Conclusions: Therefore, we propose this adaptive LC-MS/MS method as a suitable method for accurate determination of hepcidin-25 in clinical samples and as a major tool contributing to the clinical diagnosis, follow-up and management of iron-related disorders. It also opens new avenues to measure hepcidin in animal models without interspecies antigenic limitations.
Acknowledgments
We thank Aline Cheize from Waters for helping in the development of LC-MS/MS and reading the paper, Boualem Moulouel for providing ZIGI samples. Assistance Publique des Hôpitaux de Paris (AP-HP), INSERM and Paris Diderot University, France supported this work. Part of this work is funded by the program “University of Sorbonne Paris Cité, Excellence Initiative, IDEX”, reference KIRON. Dounia Houamel was supported by the Université Paris Diderot, the Société Française d’Hématologie and by the Laboratory of excellence, GR-Ex, Paris, France. The labex GR-Ex, reference ANR-11-LABX-0051 is funded by the program “Investissements d’avenir” of the French National Research Agency, reference ANR-11-IDEX-0005-02.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Supplemental Material
The online version of this article (DOI: 10.1515/cclm-2014-1093) offers supplementary material, available to authorized users.
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