Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. However, to date, no reliable and practical method for measuring the bioactive form of hepcidin in serum has been developed. Here, we used surface-enhanced laser desorp-

Histological and functional changes of the lacrimal gland might be reflected in proteomic patterns in tear fluids. In this study, we carried out a determination of the disease biomarkers in tear fluid for Sjögren's syndrome (SS) and a performance of noninvasive diagnostic test based on the proteomic patterns. Thirty-one SS patients and 57 control subjects were enrolled to this study. Their details were 23 cases with primary SS, 8 with secondary SS, 14 with dry eyes, 22 with miscellaneous ocular diseases, and 21 of healthy volunteers. Protein profiling in tear fluids was identified by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Multiple protein changes were reproducibly detected in the primary SS group, including 10 potential novel biomarkers. Seven of the biomarkers (2094, 2743, 14191, 14702, 16429, 17453, 17792 m/z) were down-regulated and 3 biomarkers (3483, 4972, 10860 m/z) were up-regulated in primary SS group, comparing to the protein profiles of control subjects. When cutoff value of SS down-score was set less than 0.5, this result yielded 87% sensitivity and 100% specificity. The positive predictive value for this sample set was 100%. There was a significant inverse correlation between SS down-scores and epithelial damages of the ocular surface in primary SS patients. These findings support the potential of proteomic pattern technology in tear fluids as the noninvasive diagnostic test for primary SS.

The relationship between serum hepcidin, a key regulator of body iron homeostasis, and erythropoiesis was investigated before and after stem cell transplantation in 31 patients with hematopoietic malignancies. Serum hepcidin-25 was monitored using a liquid chromatography-tandem mass spectrometry-based assay system. Other iron-and erythropoiesis-related parameters and known hepcidin regulators, such as interleukin-6 and growth differentiation factor-15, were also monitored. The serum hepcidin level peaked one week after stem cell transplantation, followed by a gradual decrease with a parallel change in interleukin-6 and a reciprocal change in reticulocyte count. Multivariate regression analysis demonstrated that the serum hepcidin level at four weeks after stem cell transplantation showed significant inverse correlations with erythropoietic activity markers, such as the soluble transferrin receptor, but not with growth differentiation factor-15. These results indicate the existence of an unknown functional erythropoiesis-associated circulating factor, other than growth differentiation factor-15, that negatively regulates hepcidin production in stem cell transplantation settings.

No 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.