Human hepcidin acts by binding to the plasma membrane iron exporter ferroportin. Binding results in the internalization and degradation of the complex and reduction of plasma iron levels. Because of its central role in the regulation of plasma iron levels, hepcidin has emerged as a leading analytical target related to multiple disorders including, the anemias of chronic inflammation, aging, cancer and autoimmune disorders as well as iron loading in hereditary hemochromatosis and transfusion-dependent anemias. Although early studies have used both SELDI-TOF and LC-MS/MS technology with some success, to date, very limited data is available for urine and serum correlation, normal vs. disease reference ranges, and gender-based or age-based reference ranges.

Methods:

Serum samples from healthy individuals (n=100) were analyzed to determine reference intervals for human hepcidin 25. Additionally, matched urine and serum samples (n=140) were analyzed to determine hepcidin correlation in different sample types. Minimal sample preparation was performed using a 200 uL aliquot acidified with 10% Formic acid, spiked with isotope labeled hepcidin 25 internal standard, and processed by ultracentrifugation (Millipore). Two- dimensional chromatography was used to enrich and separate hepcidin 25 prior to mass detection using the API 4000 QTrap mass spectrometer (Applied Biosystems/ SCIEX). The following m/z transitions were monitored for hepcidin 25; 558.4 to 693.8 and 698.3 to 1040.1. Isotope labeled internal standard was monitored by 563.5 to 625.4 and 703.7 to 862.9. Three points calibration (1, 10 and 100 ng/mL) was used for simultaneous quantitation of hepcidin in both urine and serum in a 4 minutes analysis.

Results:

A total of 240 samples were analyzed to determine both healthy reference intervals and serum/urine correlation for human hepcidin 25. The limit of detection (LOD) for the assay was 0.1 ng/mL with an upper limit of linearity (ULOL) of 1000 ng/mL. Extraction recovery was greater than 85%, with quantitation accuracy averaging greater than 95%. The calculated reference ranges for hepcidin 25 in serum was 2 to 35 ng/mL and in urine 1 to 107 ng/mL. The serum/urine correlation for hepcidin 25 was y = 0.032x + 8.43 and r =0.105. Within run CV% was less than 7% and between run CV% less than 11%.

Conclusions:

Knowledge to monitor the level of Hepcidin and accurate calculated reference intervals in urine and serum may help physicians to control and trouble shoot most of the iron deficiency and anemic diseases. Hepcidin quantization with this method would open a new window to provide solutions for iron deficiency issues.

(Supported by ARUP Institute for Clinical and Experimental Pathology)