Abstract
Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25+40 as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.
Highlights
The peptide hormone hepcidin plays a central role in regulating dietary iron absorption and body iron distribution
Due to the slightly different biochemical characteristics (Table 1), the behavior of both peptides appeared different in the IMAC-Cu2+ SELDI-TOF approach, which motivated us to set-up hepcidin measurements using a weak cation exchange (WCX-)MALDI approach in which both peptides performed exactly the same
An important incentive of the present study was to design a broadly applicable heavy hepcidin-25 peptide that can be used as internal standard in different assay formats on various mass spectrometry platforms as this might contribute to the harmonization of the different hepcidin assays throughout the world [25]
Summary
The peptide hormone hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. Under physiological conditions N2terminal truncated hepcidin220 and 222 peptides have been observed in the urine, but not or at low concentrations, in plasma [2,3,4,5,6,7] These smaller hepcidin isoforms mostly occur in plasma in diseases that are associated with significantly increased hepcidin concentrations, such as sepsis and kidney failure [6,8,9,10]. Data suggest that a calcium-independent tissue activity present in pancreas extracts might be responsible for the systemic N-terminal truncation of hepcidin-25 to hepcidin-22, and that dipeptidylpeptidase 4 is involved in the processing of hepcidin-22 into hepcidin-20 [11,12] It is unknown whether hepcidin isoforms can be the result of ex-vivo processing. This hepcidin analogue was chosen because these assays were run on low/
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