Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material

Vorm, Lisa N. van der; Hendriks, Jan C.M.; Laarakkers, Coby M.; Klaver, Siem M.; Armitage, Andrew E.; Bamberg, Alison; Geurts-Moespot, Anneke; Girelli, Domenico; Herkert, Matthias; Itkonen, Outi; Konrad, Robert J.; Tomosugi, Naohisa; Westerman, Mark; Bansal, Sukhvinder S.; Campostrini, Natascia; Drakesmith, Hal; Fillet, Marianne; Olbina, Gordana; Pasricha, Sant‐Rayn; Pitts, Kelly R.; Sloan, John H.; Tagliaro, Franco; Weykamp, Cas; Swinkels, Dorine W.

doi:10.1373/clinchem.2016.256768

Cited by 75 publications

(59 citation statements)

References 33 publications

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“…For the isotopically labeled internal standard, a determination of the isotopic purity would be helpful. Thus, the efforts towards hepcidin-25 standardization [31] could be complemented by the development of a reference material additionally characterized by qNMR, ICP-MS [49] and amino acid analysis [41]. We would like to raise the awareness that synthetic and natural peptides can contain a large number of different proteoforms, from which isomers and metal complexes are particularly difficult to identify.…”

Section: Discussionmentioning

confidence: 99%

“…This illustrates the importance of the correct and complete structure when working with a biochemical compound, and the need remains to examine the interaction of human hepcidin further. This could have a meaningful impact on hepcidin-25 assays and contribute to solving the problems associated with hepcidin-25 quantification [30,31]. Therefore, the present study investigated the complex formation of hepcidin-25 with metals based on the hypothesis that the natural, bioactive form of human hepcidin-25 contains a copper(II) ion.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Abbas

Vranić

Hoffmann

et al. 2018

IJMS

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Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Abbas

Vranić

Hoffmann

et al. 2018

IJMS

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“…Hepcidin is an acute-phase reactant (50), is suppressed in the presence of increased erythropoietic activity unrelated to iron status (51), and has a large (49%) intra-individual variability (52). Hepcidin can be measured with good reproducibility in serum or urine by mass-spectrometry assays or immunoassays, but there are considerable assay differences and no standardization yet (53). A recent inter-laboratory comparison study identified a commutable secondary reference material, which–if used as a common calibrator–could harmonize assay results to an achievable equivalence of 7.7% from the current 28.6% (53).…”

Section: Emerging Iron Status Indicatorsmentioning

confidence: 99%

Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges

Pfeiffer

Looker

2017

The American Journal of Clinical Nutrition

164

167

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Biochemical assessment of iron status relies on serum-based indicators, such as serum ferritin (SF), transferrin saturation, and soluble transferrin receptor (sTfR), as well as erythrocyte protoporphyrin (EP). These indicators present challenges for clinical practice and national nutrition surveys, and often iron status interpretation is based on the combination of several indicators. The diagnosis of iron deficiency (ID) through SF concentration, the most commonly used indicator, is complicated by concomitant inflammation. sTfR concentration is an indicator of functional ID that is not an acute-phase reactant, but challenges in its interpretation arise due to the lack of assay standardization, common reference ranges, and common cutpoints. It is unclear which indicators are best suited to assess excess iron status. The value of hepcidin, non-transferrin bound iron, and reticulocyte indices is being explored in research settings. Serum-based indicators are generally measured on fully-automated clinical analyzers available in most hospitals. Although international reference materials have been available for years, the standardization of immunoassays is complicated by the heterogeneity of antibodies used and the absence of physico-chemical reference methods to establish “true” concentrations. From 1988 to 2006, the assessment of iron status in the National Health and Nutrition Examination Survey (NHANES) was based on the multi-indicator ferritin model. However, the model did not indicate the severity of ID and produced categorical estimates. More recently, iron status assessment in NHANES has used the total body iron stores (TBI) model, in which the log ratio of sTfR to SF is assessed. Together, sTfR and SF concentrations cover the full range of iron status. The TBI model better predicts the absence of bone marrow iron than SF concentratio alone and TBI can be analyzed as a continuous variable. Additional consideration of methodologies, interpretation of indicators, and analytical standardization is important for further improvements in iron status assessment.

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“…For the isotopically labelled internal standard, a determination of the isotopic purity would be helpful. Thus, the efforts towards hepcidin-25 standardization [38] could be complemented by the development of a reference material additionally characterized by qNMR, ICP-MS [61] and amino acid analysis [53]. We would like to raise the awareness that synthetic and natural peptides can contain a large number of different proteoforms, from which isomers and metal complexes are particularly difficult to identify.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the need remains to further examine the interaction of human hepcidin with metals and supports the importance of the correct structure when working with a biochemical compound. This could have a meaningful impact on hepcidin-25 assays and contribute to solving the problems associated to hepcidin-25 quantification [37,38]. Therefore, the present study investigated the complex formation of hepcidin-25 with metals based on the hypothesis that the natural, bioactive form of human hepcidin-25 contains a copper(II) ion.…”

Section: Analysis Of Hepcidin-metal Complexesmentioning

confidence: 99%

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Abbas

Vranić

Hoffmann

et al. 2018

Preprint

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Abstract:Hepcidin-25 was identified as the main iron regulator in the human body by binding to the sole ironexporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as ATCUN (amino terminal Cu(II)-and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the formed complex of hepcidin-25 with copper could reveal insights into its biological role. The present work is mainly focused on the study of the metal-bound form of hepcidin-25, which could be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25, which was achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry MS/MS, high resolution mass spectrometry HRMS) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a 3D model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

show abstract

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material

Cited by 75 publications

References 33 publications

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

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