Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n = 50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.     

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro‐high‐performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile‐phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.     

类风湿关节炎患者血清骨形态发生蛋白6和铁调素与贫血的关系

目的探讨类风湿关节炎（RA）患者骨形态发生蛋白6(BMP-6)和铁调素(Hepc)与贫血的关系。方法采用酶联免疫吸附法测定99例RA患者、19例系统性红斑狼疮（SLE）患者、40例健康志愿者的血清BMP-6、Hepc水平,同时检测RA患者的红细胞计数（RBC）、血红蛋白水平（HGB）、白细胞介素6(IL-6)、血清铁（SI）、血清可溶性转铁蛋白受体（sTfR）、铁蛋白（SF），分析比较RA组与SLE组和健康对照组、RA患者贫血组与非贫血组、慢性病性贫血（ACD）与慢性病伴缺铁性贫血（IDA+ACD）组间及不同疾病活动度组间BMP-6、Hepc和其他贫血相关指标,以及RA患者血清BMP-6和Hepc水平与其他因素间的相关性。结果 RA患者贫血发生率为59.6%，其中ACD占52.5%, IDA+ACD占47.5%。血清BMP-6和Hepc水平在RA患者中明显升高，与SLE组、健康对照组的差异均有统计学意义（均P＜0.05）。RA贫血组的IL-6水平及类风湿关节炎疾病活动性评分（DAS28）较非贫血组均明显升高，差异有统计学意义（P＜0.05），而两者SI、SF、BMP-6和Hepc水平的差异均无统计学意义（均P＞0.05）；RA伴ACD患者的血清SF、Hepc、BMP-6水平及DAS28评分均明显高于RA伴IDA+ACD组，差异均有统计学意义（均P＜0.05）。DAS28＞5.1的RA疾病高度活动组血清BMP-6及Hepc较DAS28评分≤5.1的疾病非高度活动组明显升高，而HGB水平明显降低，差异均有统计学意义（均P＜0.05）。RA患者的血清BMP-6与Hepc水平呈正相关（P＜0.01），且两者均与IL-6、DAS28呈正相关（P＜0.05），BMP-6还与SI呈正相关（P＜0.05）。结论 RA患者贫血类型以ACD为主，其血清BMP-6和Hepc水平均明显升高，在伴ACD或疾病高度活动的RA患者中升高尤其明显，且互相关系密切，说明BMP-6和Hepc水平异常正是诱导RA患者发生ACD的主要因素之一，两者可能可以作为RA患者ACD治疗的靶点，其在RA中的作用机制值得进一步研究探索。     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Evidence for positive Darwinian selection on the hepcidin gene of Perciform and Pleuronectiform fishes

Hepcidin is a small cysteine-rich peptide that plays an important role in antimicrobial activity and in maintaining iron homeostasis in vertebrates. Here we report on the underlying mechanism that maintains high sequence diversities among the hepcidin-like variants of perciform and pleuronectiform fishes. In contrast to mammals, maximum likelihood-based codon substitution analyses revealed that positive Darwinian selection (nonsynonymous to synonymous substitution, ω > 1) is the likely cause of accelerated rate of amino acid substitutions in the hepcidin mature peptide region of these fishes. Comparison of models incorporating positive selection (ω > 1) at certain sites with models not incorporating positive selection (ω < 1) failed to reject (p = 0) the evidence of positive selection among the codon sites of percifom and pleuronectiform hepcidin. The adaptive evolution of this peptide in perciform and pleuronectiform fishes might be directed by pathogens when the host is exposed to new habitats/environments.