Measurement of urinary hepcidin levels by SELDI-TOF-MS in HFE-hemochromatosis

INTRODUCTION: Insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve the diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight(SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. This study measured urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients at diagnosis and during treatment, including both C282Y homozygous and C282Y/H63D compound heterozygotes. METHODS: We used a protocol based on PBSIIc mass spectrometer and Normal Phase chips. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy. RESULTS: C282Y homozygotes either at diagnosis, or after phlebotomy had significantly lower urinary hepcidin levels than controls(P<0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis higher than controls, while the hepcidin/ferritin ratio was significantly decreased (P<0.001) suggesting inadequate hepcidin production. After phlebotomy, mean hepcidin levels in the compound heterozygotes were significantly lower than in controls (P<0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a significant correlation (rho=0.64; P=0.024). CONCLUSIONS: This study supports the use of SELDI-TOF-MS for measuring hepcidin levels in research and clinical applications. Our results in phlebotomized patients suggest that the depletion of iron stores may further exacerbate the HFE-related hepcidin defect.