Abstract

Hepcidin, first identified in human urine as a bactericidal peptide, is now considered a central molecule that regulates iron metabolism. Synthesis of hepcidin is known to be up-regulated by at least two signals: iron signal and interleukin-6 (IL-6) signal. The existence of a third signal, an erythropoiesis-associated pathway, has recently been suggested by several researchers experimenting with a mouse model treated with either chemotherapy or irradiation. However, the finding has not yet been clarified in human clinical settings partly because of difficulty in quantitatively measuring the serum level of hepcidin. Therefore, we monitored and assessed the association between the serum level of hepcidin and erythropoietic activity before and after stem cell transplantation for hematological malignancies, because the conditioning regimen followed by SCT dramatically changes the status of erythropoiesis in patients. The serum level of hepcidin-25 was quantitatively measured using a liquid chromatography tandem mass spectrometry-based assay system with synthetic isotopic hepcidin as an internal control, which made our quantification much more reliable and reproducible with very small intra- and inter-assay CVs. (Intra-assay and inter-assay CVs were <6.7% and <8.8%, respectively.) We also measured serum levels of various iron-related parameters and IL-6 as well as bone morphogenetic protein (BMP)-2 and BMP-4. The level of serum hepcidin-25 at week -1 was high (mean ± SD; 74.9 ± 93.9 ng/ml), compared to that in control sera from 28 healthy volunteers (mean ± SD; 21.9 ± 12.3 ng/ml), and the level further increased and peaked at week+1 after SCT (mean ± SD; 255.9 ± 99.3 ng/ml), possibly due to the high level of IL-6 and suppression of erythropoiesis. The hepcidin level of patients at week+4 with delayed erythropoiesis (reticulocyte count less than 40,000 /μl) was significantly higher (p<0.02) than that in those with rapid erythropoiesis (reticulocyte count more than 80,000 /μl). The result did not change even if cases were limited to those without elevated IL-6 levels to exclude the influence of IL-6 on hepcidin expression. Transferrin saturation increased to almost 100% after conditioning therapy and slowly decreased concurrent with erythroid recovery. In contrast, the serum ferritin level tended to increase even after erythroid engraftment irrespective of serum IL-6 or hepcidin levels and the volume of blood cell transfusions after SCT. The level of serum BMP-2 was significantly elevated at all of these time points after SCT compared to that in control sera from 6 healthy volunteers. Among these findings, the level of serum hepcidin-25 was inversely and precisely correlated with reticulocyte counts during and after SCT, suggesting that serum hepcidin-25 may be a good indicator of erythropoietic activity as well as iron utilization by erythropoiesis. These findings indicated that hepcidin is regulated by erythropoiesis through a putative erythropoiesis-associated factor. High serum hepcidin levels observed in SCT patients might have contributed to the disturbance of iron homeostasis and increased serum ferritin levels. Further investigation is needed to clarify the clinical significance of hepcidin in SCT settings.

Author notes

Disclosure:Ownership Interests: N.T. doubles as President of Medical Care Proteomics Biotechnology Co. Ltd. (Ishikawa-ken, Japan). Research Funding: This work was supported in part by a grant-in aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and a grant from Takeda Science Foundation.