促红细胞生成素和人生长激素兴奋剂检测方法的研究进展

在2008年世界反兴奋剂组织颁布的兴奋剂目录中,S2项肽类激素及相关品种均属于内源性生物大分子物质,如何区分所检测物质属于外界摄入还是机体分泌是此项检测的重点与难点。本文针对其中应用最为普遍、研究较为深入的促红细胞生成素(EPO)和人生长激素(hGH)的检测,从间接血液指标检测、直接检测途径等方面进行了分类评述,侧重于从理化分析方法、免疫分析方法角度阐述识别及区分重组蛋白与内源性蛋白的新途径。     

左卡尼汀联合EPO治疗CKDⅡ～Ⅳ期肾性贫血的临床研究

目的评价左卡尼汀联合促红细胞生成素(EPO)对慢性肾衰竭(CKD)Ⅱ～Ⅳ期非透析阶段肾性贫血的疗效,并讨论联合用药的不良反应.方法将自贡市第四人民医院2008年1月—2011年12月收治的CKDⅡ～Ⅳ期肾性贫血患者78例进行随机双盲法分组,每组各39例.治疗组:每周静脉注射左卡尼汀及EPO;对照组:每周静脉注射EPO,两组EPO用量相同,疗程12周,采用回顾性分析方法统计各组患者红蛋白、红细胞压积的升高情况,EPO的用量变化和不良反应的发生率.结果治疗组和对照组相比血红蛋白、红细胞压积明显升高,EPO的用量明显减少,并发症高血压发生率显著降低.结论联合左卡尼汀能提高EPO治疗肾性贫血疗效,降低EPO的不良反应发生率.     

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 μL volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 μL eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.     

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis–mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.     

一种具有刺激红系细胞集落生成的螺旋藻（Spirulina platensis）蛋白

从螺旋藻体中通过ＤＥＡＥ纤维素层析、硫酸铵分部沉淀和葡聚糖Ｇ７５凝胶过滤得到一种具有离体情况下刺激红系细胞集落生成的功能蛋白质．ＳＤＳ—ＰＡＧＥ测分子量为１５０００道尔顿，等电点为４．８，含有藻蓝胆素．该蛋白命名为螺旋藻１５０００蛋白（Ｓｐｉｒｕｌｉａｐｌａｔｅｎｓｉｓ１５０００，ＳＰ—１５０００）．     

耐力运动员中促红细胞生成素的滥用及其检测

据信,有一些耐力运动员滥用促红细胞生成素.虽然促红细胞生成素可通过增加机体红细胞数目和血红蛋白含量,提高最大吸氧量,从而增强运动员耐力,但滥用促红细胞生成素,可能对运动员身体有很大危害.目前已有一些检测促红细胞生成素滥用的方法.然而,有着许多问题有待解决.