Binding of Hepcidin to Plasma Proteins

To the Editor:The hepcidin hormone of 25 amino acid residues is a key regulator of iron homeostasis (1). Hepcidin has been shown to bind in vitro to α2-macroglobulin (α2M)1 and albumin in human plasma (2). It is not known, however, to what extent hepcidin is bound to proteins in vivo. For purposes of clinical interpretation, it is critical to know whether protein-bound or free hepcidin is being quantified.To characterize the binding of hepcidin to plasma proteins, we used gel filtration to fractionate 0.5 mL serum samples from both healthy individuals and patients undergoing hematopoietic stem cell transplantation. We similarly fractionated mixtures of hepcidin and α2M or albumin [1 g/L in 10 mmol/L potassium phosphate buffer, pH 7.4, containing 150 mmol/L NaCl (PBS)]. We used a Superdex™ 200 10/300 GL column (GE Healthcare Biosciences) equilibrated with PBS at a flow rate 0.5 mL/min. The column was calibrated with synthetic hepcidin (Peptide Institute), human albumin, and α2M. We collected 0.5-mL fractions and measured hepcidin in the fractions by HPLC–tandem mass spectrometry (LC-MS/MS) as previously described (3). We observed a single peak, corresponding to …