Prediction of human iron bioavailability using rapid c-ELISAs for human plasma hepcidin

Nicole U. Stoffel; Christophe Zeder; Eloïse Fort; Dorine W. Swinkels; Michael B. Zimmermann; Diego Moretti

doi:10.1515/cclm-2017-0097

Article

Prediction of human iron bioavailability using rapid c-ELISAs for human plasma hepcidin

Nicole U. Stoffel , Christophe Zeder , Eloïse Fort , Dorine W. Swinkels , Michael B. Zimmermann and Diego Moretti

Published/Copyright: June 17, 2017

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 55 Issue 8

Abstract

Background:

Hepcidin is the central systemic regulator of iron metabolism, but its quantification in biological fluids is challenging. Rapid, accurate and user-friendly methods are needed. Our aim was to assess the ability of hepcidin as measured by three different c-ELISA assays to predict iron bioavailability in humans.

Methods:

The three assays used were commercially available DRG and Peninsula assays and the c-ELISA method performed at Radboud University Medical Centre, Nijmegen, The Netherlands (Hepcidinanalysis.com), validated by comparative measurements with time-of-flight mass spectrometry. We analyzed plasma samples (n=37) selected to represent a broad range of hepcidin concentrations from a subgroup of healthy, iron-depleted women in a study assessing fractional absorption from iron supplements.

Results:

In single regressions, all three c-ELISA assays were predictors of fractional iron absorption: R²=0.363 (DRG), R²=0.281 (Peninsula) and R²=0.327 (Hepcidinanalysis.com). In multiple regressions, models including hepcidin measured with either DRG-, Peninsula or Hepcidinanalysis.com explained 55.7%, 44.5% and 52.5% of variance in fractional absorption, and hepcidin was a strong predictor of fractional absorption irrespective of the hepcidin assays used. However, we found significant differences in absolute values for hepcidin between different methods. Both the DRG assay’s (y=0.61x+0.87; R²=0.873) and the Peninsula assay’s measurements (y=1.88x+0.62; R²=0.770) were correlated with Hepcidinanalysis.com.

Conclusions:

The biological variability in plasma hepcidin, (inter-sample CV) was 5–10-fold higher for both the Peninsula and DRG assay than the analytical variably (inter-run within-sample CV) suggesting substantial discriminatory power to distinguish biological hepcidin variation. Between methods, prediction of iron bioavailability in generally healthy iron depleted subjects appears comparable.

Keywords: anemia of chronic disease; hepcidin; iron absorption; iron deficiency anemia; iron overload

Acknowledgments

We would like to thank Andrew Armitage, Weatherall Institute of Molecular Medicine University of Oxford, Oxford, UK for very valuable analytical advice on the calibration of the Peninsula measurement kit.

Author contributions: NUS and DM designed the study. NUS and EF did the hepcidin measurements with the two commercially available c-ELISA assays. NUS analyzed the data and wrote the first draft of the manuscript. All authors contributed to the final version of the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: DWS is an employee of Radboud University and Medical Centre that offers high quality hepcidin measurements to the scientific, medical and pharmaceutical community, at a fee for service basis.
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.

References

1. Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001;276:7811–9.10.1074/jbc.M008923200Search in Google Scholar

2. Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003;101:2461–3.10.1182/blood-2002-10-3235Search in Google Scholar

3. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090–3.10.1126/science.1104742Search in Google Scholar

4. Girelli D, Nemeth E, Swinkels DW. Hepcidin in the diagnosis of iron disorders. Blood 2016;127:2809–13.10.1182/blood-2015-12-639112Search in Google Scholar

5. Theurl I, Aigner E, Theurl M, Nairz M, Seifert M, Schroll A, et al. Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications. Blood 2009;113:5277–86.10.1182/blood-2008-12-195651Search in Google Scholar

6. van Santen S, van Dongen-Lases EC, de Vegt F, Laarakkers CM, van Riel PL, van Ede AE, et al. Hepcidin and hemoglobin content parameters in the diagnosis of iron deficiency in rheumatoid arthritis patients with anemia. Arthritis Rheum 2011;63:3672–80.10.1002/art.30623Search in Google Scholar

7. Bregman DB, Morris D, Koch TA, He A, Goodnough LT. Hepcidin levels predict nonresponsiveness to oral iron therapy in patients with iron deficiency anemia. Am J Hematol 2013;88:97–101.10.1002/ajh.23354Search in Google Scholar

8. Moretti D, Goede JS, Zeder C, Jiskra M, Chatzinakou V, Tjalsma H, et al. Oral iron supplements increase hepcidin and decrease iron absorption from daily or twice-daily doses in iron-depleted young women. Blood 2015;126:1981–9.10.1182/blood-2015-05-642223Search in Google Scholar

9. Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, Schulz-Knappe P, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS letters 2000;480:147–50.10.1016/S0014-5793(00)01920-7Search in Google Scholar

10. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001;276:7806–10.10.1074/jbc.M008922200Search in Google Scholar PubMed

11. Kroot JJ, Laarakkers CM, Geurts-Moespot AJ, Grebenchtchikov N, Pickkers P, van Ede AE, et al. Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders. Clin Chem 2010;56:1570–9.10.1373/clinchem.2010.149187Search in Google Scholar PubMed

12. Zimmermann MB, Troesch B, Biebinger R, Egli I, Zeder C, Hurrell RF. Plasma hepcidin is a modest predictor of dietary iron bioavailability in humans, whereas oral iron loading, measured by stable-isotope appearance curves, increases plasma hepcidin. Am J Clin Nutr 2009;90:1280–7.10.3945/ajcn.2009.28129Search in Google Scholar PubMed

13. Erhardt JG, Estes JE, Pfeiffer CM, Biesalski HK, Craft NE. Combined Measurement of Ferritin, Soluble Transferrin Receptor, Retinol Binding Protein, and C-Reactive Protein by an Inexpensive, Sensitive, and Simple Sandwich Enzyme-Linked Immunosorbent Assay Technique. J Nutr 2004;134:3127–32.10.1093/jn/134.11.3127Search in Google Scholar PubMed

14. Pasricha SR, Atkinson SH, Armitage AE, Khandwala S, Veenemans J, Cox SE, et al. Expression of the iron hormone hepcidin distinguishes different types of anemia in African children. Sci Transl Med. 2014;6:235re3.10.1126/scitranslmed.3008249Search in Google Scholar PubMed

15. Wegmuller R, Bah A, Kendall L, Goheen MM, Mulwa S, Cerami C, et al. Efficacy and safety of hepcidin-based screen-and-treat approaches using two different doses versus a standard universal approach of iron supplementation in young children in rural Gambia: a double-blind randomised controlled trial. BMC Pediatr 2016;16:149.10.1186/s12887-016-0689-4Search in Google Scholar PubMed PubMed Central

16. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986;i:307–10.10.1016/j.ijnurstu.2009.10.001Search in Google Scholar

17. Roe MA, Collings R, Dainty JR, Swinkels DW, Fairweather-Tait SJ. Plasma hepcidin concentrations significantly predict interindividual variation in iron absorption in healthy men. Am J Clin Nutr 2009;89:1088–91.10.3945/ajcn.2008.27297Search in Google Scholar PubMed

18. Young MF, Glahn RP, Ariza-Nieto M, Inglis J, Olbina G, Westerman M, et al. Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young women. Am J Clin Nutr 2009;89:533–8.10.3945/ajcn.2008.26589Search in Google Scholar PubMed

19. Cusick SE, Opoka RO, Abrams SA, John CC, Georgieff MK, Mupere E. Delaying Iron Therapy until 28 Days after Antimalarial Treatment Is Associated with Greater Iron Incorporation and Equivalent Hematologic Recovery after 56 Days in Children: A Randomized Controlled Trial. J Nutr 2016;146:1769–74.10.3945/jn.116.233239Search in Google Scholar PubMed PubMed Central

20. Prentice AM, Doherty CP, Abrams SA, Cox SE, Atkinson SH, Verhoef H, et al. Hepcidin is the major predictor of erythrocyte iron incorporation in anemic African children. Blood 2012;119:1922–8.10.1182/blood-2011-11-391219Search in Google Scholar PubMed PubMed Central

21. van der Vorm LN, Hendriks JC, Laarakkers CM, Klaver S, Armitage AE, Bamberg A, et al. Toward worldwide hepcidin assay harmonization: identification of a commutable secondary reference material. Clin Chem 2016;62:993–1001.10.1373/clinchem.2016.256768Search in Google Scholar PubMed

22. Kroot JJC, Kemna EHJM, Bansal S, Busbridge M, Campostrini N, Girelli D, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin: Need for standardization. Blood 2008;112:50–1.10.1182/blood.V112.11.120.120Search in Google Scholar

23. Kroot JJC, van Herwaarden AE, Tjalsma H, Jansen RTP, Hendriks JCM, Swinkels DW. Second round robin for plasma hepcidin methods: First steps toward harmonization. Am J Hematol 2012;87:977–83.10.1002/ajh.23289Search in Google Scholar PubMed

24. Wray K, Allen A, Evans E, Fisher C, Premawardhena A, Perera L, et al. Hepcidin detects iron deficiency in Sri Lankan adolescents with a high burden of hemoglobinopathy: A diagnostic test accuracy study. Am J Hematol 2017;92:196–203.10.1002/ajh.24617Search in Google Scholar PubMed PubMed Central

25. Peslova G, Petrak J, Kuzelova K, Hrdy I, Halada P, Kuchel PW, et al. Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood. Blood 2009;113:6225–36.10.1182/blood-2009-01-201590Search in Google Scholar PubMed

26. Itkonen O, Stenman UH, Parkkinen J, Soliymani R, Baumann M, Hamalainen E. Binding of hepcidin to plasma proteins. Clin Chem 2012;58:1158–60.10.1373/clinchem.2012.186916Search in Google Scholar PubMed

27. Crowther JR. The ELISA Guidebook. Second Edition. Totowa, NJ, USA: Springer Protocols, 2009.10.1007/978-1-60327-254-4Search in Google Scholar

28. Roe MA, Spinks C, Heath AL, Harvey LJ, Foxall R, Wimperis J, et al. Serum prohepcidin concentration: no association with iron absorption in healthy men; and no relationship with iron status in men carrying HFE mutations, hereditary haemochromatosis patients undergoing phlebotomy treatment, or pregnant women. Br J Nutr 2007;97:544–9.10.1017/S0007114507336829Search in Google Scholar PubMed

Received: 2017-2-1

Accepted: 2017-4-25

Published Online: 2017-6-17

Published in Print: 2017-7-26

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Articles in the same Issue

https://doi.org/10.1515/cclm-2017-0097

Keywords for this article

anemia of chronic disease; hepcidin; iron absorption; iron deficiency anemia; iron overload