Regulation of serum hepcidin levels in sickle cell disease

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Intra-individual variability of serum hepcidin-25 in haemodialysis patients using mass spectrometry and ELISA

Background. Measurement of serum hepcidin levels may provide a useful alternative to the current methods of determining iron status in chronic haemodialysis (HD) patients. However, the biological variability of this pivotal regulator of iron homeostasis is unclear, and the impact of inflammation, dialysis clearance and iron therapy on hepcidin variability has not been established.Methods. Two independent studies in chronic HD patients were conducted; serum hepcidin levels were measured at the start of dialysis sessions in 20 UK patients and in 43 Dutch patients by mass spectrometry (MS). Samples from UK patients were also analysed by a competitive enzyme-linked immunosorbent assay (cELISA). Coefficient of variance (CV1) was calculated and potential factors affecting CV1 were also examined.Results. The median CV1 (inter-quartile range) was 23% (17-28) for the UK MS, 26% (17-48) for the Dutch MS and 23% (17-39) for the UK cELISA. The CV1 was similar in those patients receiving and those not receiving regular intravenous iron. The CV1 was not associated with the degree of inflammation. Hepcidin levels were higher following an inter-dialytic period of 3 versus 2 days (P = 0.02).Conclusions. These findings suggest considerable variability of serum hepcidin levels in HD patients. Inflammation and the use of iron did not impact on the degree of variability, and hepcidin levels were higher after an inter-dialytic period of 3 versus 2 days. These findings need to be taken into account in future studies assessing the utility of serum hepcidin as a guide to the use of iron or erythropoiesis-stimulating agents therapy.</p

Provisional standardization of hepcidin assays:Creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95\ub10.11 nmol/L) and middle concentration (3.75\ub10.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin

Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

Contains fulltext : 117320.pdf (publisher's version ) (Closed access)Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75mg/kg bw DF by oral gavage and 24h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p<0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p<0.001 and p<0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p<0.001) and protein expression of PCNA (p<0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury