Unraveling Hepcidin Plasma Protein Binding: Evidence from Peritoneal Equilibration Testing

Peptide hormone hepcidin regulates systemic iron metabolism and has been described to be partially bound to α2-macroglobulin and albumin in blood. However, the reported degree of hepcidin protein binding varies between <3% and ≈89%. Since protein-binding may influence hormone function and quantification, better insight into the degree of hepcidin protein binding is essential to fully understand the biological behavior of hepcidin and interpretation of its measurement in patients. Here, we used peritoneal dialysis to assess human hepcidin protein binding in a functional human setting for the first time. We measured freely circulating solutes in blood and peritoneal fluid of 14 patients with end-stage renal disease undergoing a peritoneal equilibration test to establish a curve describing the relation between molecular weight and peritoneal clearance. Calculated binding percentages of total cortisol and testosterone confirmed our model. The protein-bound fraction of hepcidin was calculated to be 40% (±23%). We, therefore, conclude that a substantial proportion of hepcidin is freely circulating. Although a large inter-individual variation in hepcidin clearance, besides patient-specific peritoneal transport characteristics, may have affected the accuracy of the determined binding percentage, we describe an important step towards unraveling human hepcidin plasma protein binding in vivo including the caveats that need further research.