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 free hepcidin, eluting at 21–25 mL. In patient sera containing 36.6 nmol/L (102 μg/L) and 50.4 nmol/L (141 μg/L) endogenous hepcidin, LC-MS/MS analysis revealed very small hepcidin peaks in fractions containing α2M and albumin, respectively (Table 1), but the hepcidin concentration was below the limit of quantification (3). The hepcidin peak area corresponded to 0.7% and 0.9% of the main hepcidin peak, respectively, in the α2M fractions and to 0.9% and 1.3% of the main hepcidin peak, respectively, in the albumin fractions. Thus, approximately 98% of hepcidin was free in samples with increased hepcidin. This result contrasts with the findings of Peslova et al., who calculated that only 11% of hepcidin in the circulation was free (2). The molar ratio of α2M to hepcidin varied from 0.2:1 to 5:1 in their in vitro study, whereas the mean molar ratio of α2M to hepcidin in the serum of healthy individuals is 620:1. The much higher proportion of hepcidin relative to the α2M concentration that these investigators used can explain the difference between our results and those of Peslova et al. Hepcidin was not detectable in the α2M and albumin fractions of healthy individuals.