Measurement of Urinary Hepcidin Levels by SELDI-TOF-MS in HFE-Hemochromatosis.

Introduction: An 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 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. Until now, urinary hepcidin has been measured by SELDI-TOF-MS only in small groups of C282Y homozygous patients, the majority of them under phlebotomy treatment. No data are available on C282Y/H63D compound heterozygotes, that can develop a milder clinical form of HH. This study was aimed to measure urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients.

Methods: We used a protocol based on PBSIIc mass spectromer and Normal Phase chips similar to that recently proven successful for semi-quantitative detection of urinary hepcidin. 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 (at least 30 days from last phlebotomy).

Results: C282Y homozygotes had significantly lower urinary hepcidin levels vs. controls either at diagnosis, or after phlebotomy (P < 0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis similar to controls, while the hepcidin:ferritin ratio was significantly decreased (P < 0.001) suggesting a relatively inappropriate hepcidin production. Moreover, also in this group means hepcidin levels after phlebotomy 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 good correlation (r= 0.77; P<0.0001).

Conclusions: SELDI-TOF-MS assay is confirmed to be a potentially useful tool for measuring hepcidin levels in HH. The very low hepcidin levels observed in both genotypes after phlebotomy, may suggest that iron depletion as currently achieved by standard protocols may not be the best therapy from a pathophysiological standpoint.