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Proteomics and Protein Markers |
1 Department of Clinical Chemistry, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
2 Ciphergen Biosystems, Inc., Fremont, CA.
aAddress correspondence to this author at: Department of Clinical Chemistry 441, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31 (0) 24-3541743; e-mail: e.kemna{at}akc.umcn.nl.
Background: Discovery of the central role of hepcidin in body iron regulation has shed new light on the pathophysiology of iron disorders. Information is lacking on newer analytical approaches to measure hepcidin in serum and urine. Recent reports on the measurement of urine and serum hepcidin by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) necessitate analytical and clinical evaluation of MS-based methodologies.
Methods: We used SELDI-TOF MS, immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, we investigated analytical reproducibility and biological and preanalytical variation for both serum and urine on Normal Phase 20 and Immobilized Metal Affinity Capture 30 ProteinChip arrays. We obtained samples from healthy controls and patients with documented iron-deficiency anemia, inflammation-induced anemia, thalassemia major, and hereditary hemochromatosis.
Results: Proteomic techniques showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. The interarray CV was 15% to 27%, and interspot CV was 11% to 13%. Preliminary studies showed that hepcidin-25 differentiated disorders of iron metabolism. Urine hepcidin is more affected by multiple freeze-thaw cycles and storage conditions, but less influenced by diurnal variation, than is serum hepcidin.
Conclusion: SELDI-TOF MS can be used to measure hepcidin in both serum and urine, but serum requires a standardized sampling protocol.
The following articles in journals at HighWire Press have cited this article:
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  J. J.C. Kroot, C. M.M. Laarakkers, E. H.J.M. Kemna, B. J. Biemond, and D. W. Swinkels Regulation of serum hepcidin levels in sickle cell disease Haematologica, June 1, 2009; 94(6): 885 - 887. [Full Text] [PDF]
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 M. A Roe, R. Collings, J. R Dainty, D. W Swinkels, and S. J Fairweather-Tait Plasma hepcidin concentrations significantly predict interindividual variation in iron absorption in healthy men Am. J. Clinical Nutrition, April 1, 2009; 89(4): 1088 - 1091. [Abstract] [Full Text] [PDF]
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 J. P. Karl, H. R. Lieberman, S. J. Cable, K. W. Williams, E. L. Glickman, A. J. Young, and J. P. McClung Poor Iron Status Is Not Associated with Overweight or Overfat in Non-Obese Pre-Menopausal Women J. Am. Coll. Nutr., February 1, 2009; 28(1): 37 - 42. [Abstract] [Full Text] [PDF]
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M. F Young, R. P Glahn, M. Ariza-Nieto, J. Inglis, G. Olbina, M. Westerman, and K. O O'Brien Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young women Am. J. Clinical Nutrition, February 1, 2009; 89(2): 533 - 538. [Abstract] [Full Text] [PDF]
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D. M Frazer and G. J Anderson Hepcidin compared with prohepcidin: an absorbing story Am. J. Clinical Nutrition, February 1, 2009; 89(2): 475 - 476. [Full Text] [PDF]
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 E. L. Unger, C. J. Earley, and J. L. Beard Diurnal cycle influences peripheral and brain iron levels in mice J Appl Physiol, January 1, 2009; 106(1): 187 - 193. [Abstract] [Full Text] [PDF]
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 T. Ganz, G. Olbina, D. Girelli, E. Nemeth, and M. Westerman Immunoassay for human serum hepcidin Blood, November 15, 2008; 112(10): 4292 - 4297. [Abstract] [Full Text] [PDF]
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 J. F. Collins, M. Wessling-Resnick, and M. D. Knutson Hepcidin Regulation of Iron Transport J. Nutr., November 1, 2008; 138(11): 2284 - 2288. [Abstract] [Full Text] [PDF]
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 C. Adlbrecht, S. Kommata, M. Hulsmann, T. Szekeres, C. Bieglmayer, G. Strunk, G. Karanikas, R. Berger, D. Mortl, K. Kletter, et al. Chronic heart failure leads to an expanded plasma volume and pseudoanaemia, but does not lead to a reduction in the body's red cell volume Eur. Heart J., October 1, 2008; 29(19): 2343 - 2350. [Abstract] [Full Text] [PDF]
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J. Kanda, C. Mizumoto, H. Kawabata, H. Tsuchida, N. Tomosugi, K. Matsuo, and T. Uchiyama Serum hepcidin level and erythropoietic activity after hematopoietic stem cell transplantation Haematologica, October 1, 2008; 93(10): 1550 - 1554. [Abstract] [Full Text] [PDF]
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 U. Kobold, T. Dulffer, M. Dangl, A. Escherich, M. Kubbies, R. Roddiger, and J. A. Wright Quantification of Hepcidin-25 in Human Serum by Isotope Dilution Micro-HPLC-Tandem Mass Spectrometry Clin. Chem., September 1, 2008; 54(9): 1584 - 1586. [Full Text] [PDF]
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F. Guillem, S. Lawson, C. Kannengiesser, M. Westerman, C. Beaumont, and B. Grandchamp Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency Blood, September 1, 2008; 112(5): 2089 - 2091. [Abstract] [Full Text] [PDF]
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 D. W. Swinkels and J. F. M. Wetzels Hepcidin: a new tool in the management of anaemia in patients with chronic kidney disease? Nephrol. Dial. Transplant., August 1, 2008; 23(8): 2450 - 2453. [Full Text] [PDF]
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N. C. Andrews Forging a field: the golden age of iron biology Blood, July 15, 2008; 112(2): 219 - 230. [Full Text] [PDF]
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E. H.J.M. Kemna, H. Tjalsma, H. L. Willems, and D. W. Swinkels Hepcidin: from discovery to differential diagnosis Haematologica, January 1, 2008; 93(1): 90 - 97. [Abstract] [Full Text] [PDF]
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B. A. C. van Dijk, E. H. J. M. Kemna, H. Tjalsma, S. M. Klaver, E. T. G. Wiegerinck, J.-P. Goossens, P. H. Th. J. Slee, M. H. Breuning, and D. W. Swinkels Effect of the new HJV-L165X mutation on penetrance of HFE Blood, June 15, 2007; 109(12): 5525 - 5526. [Full Text] [PDF]
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