Determination of Serum Ferritin Glycosylation in Hyperferritinemia Associated to Iron Overload and Inflammation.

BACKGROUND: Serum ferritin is a commonly used clinical biochemical parameter and hyperferritinemia is used as a surrogate marker for iron overload, acute or chronic inflammation, malignancy or cell death. The aim of the present study was to develop purification strategies of ferritin from sera to determine if micro-heterogeneity of serum ferritin can be used to differentiate the underlying cause of the hyperferritinemia. PATIENTS, MATERIALS AND METHODS: Sera from patients with hemochromatosis, rheumatologic diseases, aceruloplasminemia, ferroportin disease or iron loading anemia have been collected and stored and ferritin purified by negative affinity followed by ion exchange and size exclusion chromatography. Purified serum ferritin was analyzed by western blotting and MALDI TOF mass spectrometry and the spectra compared with the results from ferritin isolated from human liver, spleen and placenta. RESULTS: By Western blotting a major band of 19kD has been found in most sera, suggesting that the L-ferritin is the predominant isoform present in serum regardless of the cause of hyperferritinemia. Multistep chromatography can be used for significant enrichment and purification of ferritin from serum, which can be further analyzed by MALDI TOF MS. Tryptic digestion and peptide mass finger-printing by MALDI TOF MS of ferritin purified from human tissues shows differential spectra. DISCUSSION AND CONCLUSIONS: Analysis of ferritin micro-heterogeneity by MALDI TOF allows determination of the tissue origin of ferritin, which could be applied in the differential diagnostic workup of hyperferritinemia.