Analytical and biological assessment of circulating human erythroferrone

Background: Erythroferrone (ERFE) is an erythroid hormone putatively involved in stress erythropoiesis. Its regional clearance and circulating form in humans, as well as levels in normal health and coronary disease remain unclear. Methods: To establish a reference interval, ERFE was measured in 155 healthy volunteers using the Intrinsic LifeSciences ELISA. To identify trans-organ gradients in ERFE, regional blood sampling was undertaken in patients (n = 13) undergoing clinically indicated cardiac catheterisation. The Intrinsic ELISA was assessed for reproducibility, stability, linearity and possible cross-reactivity, interference and anticoagulant effects. Circulating forms of ERFE were evaluated by HPLC. Results: In healthy individuals, the median concentration of ERFE was 0.51 ng/mL (IQR: 0.12-1.25), with men (n = 78) having higher levels than women (n = 77) (0.67 vs 0.32 ng/mL, p = 0.0001). ERFE concentrations in trans-organ sampling revealed no clear organ of clearance or production. Samples with high endogenous ERFE levels were suppressed by haemoglobin (>= 2 g/L), bilirubin (>= 200 mu mol/L), lipaemia (>1 g/L), and freeze thawing (>= 2 cycles), but this was not observed with low ERFE concentrations. Endogenous ERFE immunoreactivity was 46% higher in EDTA plasma compared with serum and lithium heparin plasma. On SE-HPLC, ERFE eluted as intact and cleaved forms. Conclusion: We provide a useful reference range for ERFE in EDTA plasma. We found no specific site of secretion or clearance. The Intrinsic ELISA performed adequately but is limited by interference and stability when endogenous levels are high. Circulating forms are multiple and complex.