Novel urine hepcidin assay by mass spectrometry

Abstract

The hepatic peptide hormone hepcidin is the central regulator of iron metabolism and mediator of anemia of inflammation. To date, only one specific immuno-dot assay to measure hepcidin in urine had been documented. Here we report an alternative approach for quantification of hepcidin in urine by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Peptide peaks were detected corresponding to the 3 forms of hepcidin normally found in urine. The identity of the peptide peak equivalent to hepcidin-25 was confirmed using synthetic human hepcidin-25. Validation of our MS data on samples with various hepcidin levels showed a strong correlation with previous immuno-dot assay results (Spearman R ‫؍‬ 0.9275, P < .001). Most importantly, this hepcidin assay clearly discriminates between relevant clinical iron disorders. In conclusion, this novel MS urine hepcidin assay is easy to perform and available to a wide audience. This enables the implementation of hepcidin measurements in large clinical studies. ( IntroductionHepcidin is a small, cysteine-rich cationic peptide produced by hepatocytes, 1-3 secreted into plasma, and excreted in urine. Hepcidin expression is induced by iron stores and inflammation 3 and suppressed by hypoxia and anemia. 4 Hepcidin is proposed to be the key regulator of iron metabolism and its discovery has changed our understanding of the pathophysiology of iron disorders. It now appears that hepcidin deficiency is the cause of most types of hereditary hemochromatosis and that hepcidin excess mediates anemia of inflammation. 5 Measurements of hepcidin concentrations could therefore be useful in diagnosis of iron disorders and would provide further insight into hepcidin regulation in vivo. However, assays for hepcidin detection and quantification in plasma or urine have not been generally available, and the development of reagents has been hampered by technical difficulties. 6,7 The development of immunochemical methods based on the production of specific antihepcidin antibodies is difficult due to the small size of hepcidin (25 amino acids), conservation between animal species, 8 and the limited availability of the antigen, as the production of synthetic hepcidin in its native conformation 9 or the isolation of hepcidin from urine 2 involves complex, time-consuming procedures. To date, only one immunochemical assay was successfully used to quantify urinary hepcidin in clinical studies. 10 We sought to develop a more widely available, high-throughput assay. Here we report a new quantification method for hepcidin in urine by the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). This provides a fast assay with increased simplicity and accessibility that circumvents the difficulties encountered with antibody and antigen production. Moreover, this assay has also the potential to provide insight in the proportional contribution of the 3 known hepcidin isoforms that can be found in urine (hepcidin-20, -22, and -25)....

Molecular Control of Iron Transport

“…Although further studies are necessary to clarify this issue, it seems that hepcidin is regulated predominantly by the concentrations of its mRNA so that the conclusions from mRNA-based mouse studies and peptide-based human studies are very similar. More recently, semiquantitative assays that were based on mass spectrometry were developed for human urinary (25) and serum (26) hepcidin, and these hold promise especially for measurements of elevated hepcidin concentrations.…”

Current Status of the Measurement of Blood Hepcidin Levels in Chronic Kidney Disease

“…SELDI was the first MS method used for the semiquantitative measurement of hepcidin in urine (29) and plasma (30). Hepcidin determination in the serum of hemodialysis patients was carried out using SELDI (Ciphergen Biosystems, Freemont, CA), with a reported detection limit of 53 ng/ml (30).…”

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

“…Attempts to produce correctly folded synthetic hepcidin have proved difficult because the sequence contains eight cysteine residues that constrain the hepcidin molecule in a hairpin structure (Hunter et al 2002). Hepcidin in urine has also been quantified using mass spectrometry methods (Kemna et al 2005;Liang et al 2006;Tomosugi et al 2006).Prohepcidin, the sixty-amino-acid product of cleavage of the signal peptide from the hepcidin precursor, is expressed at the basolateral membrane domain of hepatocytes and is found in blood (Kulaksiz et al 2004). Serum prohepcidin concentrations are significantly lower in patients with hereditary haemochromatosis compared to healthy control subjects (Kulaksiz et al 2004), increase with declining kidney function (Taes et al 2004), and are correlated with haematocrit in chronic haemodialysis patients (Hsu et al 2006), but it is currently unclear whether * Corresponding author: Susan Fairweather-Tait, fax þ 44 1603 452578, email sue.fairweather-tait@bbsrc.ac.uk Abbreviations: DMT1, divalent metal transporter; sTfR, soluble transferrin receptor; LREC, local research ethics committee.…”