环氧乙烷-环氧丙烷无规共聚醚链结构的核磁共振研究

本文用NMR波谱方法研究了无规共聚醚(EPO)的链结构。首先对EPO的~1H和~(13)CNMR谱进行了讨论。并从序列结构随环氧丙烷(PO)含量的改变,分析了EPO的二元组序列和三元组序列结构,从~(13)C实验谱中归纳出EPO的取代基参数(SCS),清楚地揭示了EPO的~(13)C谱,SCS参数和序列结构之间的关系,在此基础上计算了EPO系列共聚物的序列分布和数均序列长度。     

PP／EPO共混体系混容性与结晶结构

WAXD方法测定表明PP/EPO共混体系的结晶度随EPO组份含量的增加而降低,晶胞参数与组份比无关,并计算了第二类晶格畸变。根据PP/EPO的DSC和扭辫实验得出在所有组成下PP/EPO共混体系是不相容的。但在非晶区可能部分相容。     

基因兴奋剂检测的现状与策略

本文综述了基因兴奋剂检测的现状和反基因兴奋剂的策略。归纳了可能被运动员滥用的基因兴奋剂,分析了由这些基因表达的促红细胞生成素(EPO)、人生长激素(hGH)等蛋白的检测进展,讨论了未来检测基因兴奋剂的可能策略。     

低密度聚乙烯/乙丙烯三元共聚(LDPE/EPO)共混体系的结晶动力学

用DSC方法研究了LDPE/EPO共混体系的等温及非等温结晶动力学,对LDPE/EPO共混体系的等温结晶动力学研究表明,共混物是三维生长的异相成核,共混物在各个结晶温度下的结晶过程都是以方式K_g(Ⅱ)进行的.采用联系Avrami方程和Ozawa方程导出的新非等温结晶动力学方程,处理了LDPE/EPO共混体系,得到了非等温结晶过程的一些基本参数,新方程很好地描述了此共混体系的非等温结晶动力学过程.     

兴奋剂重组人促红细胞生成素及其类似物检测方法的研究进展

重组人促红细胞生成素(rhEPO)是一种激素类兴奋剂,近年来被滥用在一些耐力性比赛项目中。由于重组与内源性EPO的氨基酸序列相同,区别很小,并且在尿样或血样中的浓度低,代谢快,给检测带来了很大的难度。本文从直接方法和间接方法两个方面综述了近几年来兴奋剂rhEPO及其类似物检测的研究进展,并结合本小组的工作展望了rhEPO检测的发展方向。     

低密度聚乙烯/乙丙烯三元共聚(LDPE/EPO)共混体系的相容性及其结晶结构

DDV、DSC、WAXD、萃取和Raman光谱实验表明,在所有组成下,LDPE/EPO共混体系共晶相容.WAXD法测定表明,LDPE/EPO共混体系的结晶度随EPO组分含量的增加而降低,EPO未能进入LDPE晶胞中.     

低密度聚乙烯/乙丙烯三元共聚(LDPE/EPO)共混体系的相容性及其结晶结构

 DDV、DSC、WAXD、萃取和Raman光谱实验表明,在所有组成下,LDPE/EPO共混体系共晶相容.WAXD法测定表明,LDPE/EPO共混体系的结晶度随EPO组分含量的增加而降低,EPO未能进入LDPE晶胞中.     

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。     

地黄提取物改善贫血大鼠记忆及其机制

地黄滋阴补血填髓,广泛用于贫血和脑疾病患者,但其功效机制尚未阐明.本文探讨地黄水提物(Rehmannia glutinosa’s water extracts,RGWE)改善血虚大鼠记忆及其滋阴补血填髓的可能机制.采用断尾放血结合注射环磷酰胺制备贫血大鼠模型,随机分溶媒组和地黄水提物3,6,10g/kg治疗组,灌服等体积自来水或不同剂量地黄水提物10天,采用Morris水迷宫观测大鼠空间记忆能力,采用酶联免疫吸附测定方法(Enzyme linked immunosorbent assay,ELISA)检测血浆红细胞生成素(EPO)水平,免疫组化和免疫蛋白印迹技术(Western blotting)检测分析脑EPO及其受体表达水平.成功制备血虚模型,表现为大鼠红细胞数和血红蛋白显著下降,与造模前比较差异显著(P<0.05),溶媒组空间记忆能力显著下降,地黄治疗组空间记忆能力明显提高,逃避潜伏期明显缩短(P<0.05),一定时间内穿越平台次数显著增多(P<0.05);免疫组化和Western blotting结果显示脑组织EPO及其受体表达、血浆EPO水平均较溶媒组明显升高(P<0.05).地黄水提物显著提高贫血...     

中国人促红细胞生成素基因组的克隆及其在COS-7细胞中的表达

本文利用PCR技术,从正常人胎肝染色体DNA库中分离克隆了长度分别为506bp,465bp的中国人促红细胞生成素(EPO)基因组片段。506bp基因片段包括外显子2(信号肽),内含子2和外显子3;465bp片段包括外显子4,内含子4和外显子5。通过在引物中设置的酶切位点将两个克隆片段进行了正确的拼接,从而得到了约1.0kb的EPO次全基因组片段,它包括除信号肽中第2,3,4位氨基酸外的所有编码区及两个内含子序列。 所克隆的次全EPO基因组插入表达载体pSV2-dhfr中的不同克隆位点,构建了3种不同的转移载体质粒,分别转染导入COS-7细胞后,3种转移载体质粒转染的细胞上清液都有明显的EPO活性。本工作证明仅含有内含子2,4序列,去除负调控区和氧敏感区序列的人次全EPO基因组可以在COS-7细胞中获得表达。     

Procedures for monitoring recombinant erythropoietin and analogues in doping control

The present report summarizes the main analytical strategies developed to identify the presence of recombinant erythropoietin (EPO) administered as a doping agent. Indirect evidence is based on the analysis of blood parameters (haemoglobin, haematocrit, reticulocytes, macrocytes, etc.) and serum markers (concentration of EPO and serum transferrin receptors, etc.). The problem of intertechnique comparison for reliable results evaluation is emphasized, especially for serum markers. Charge differences between isoforms of recombinant EPO and native urinary EPO are the grounds for the isoelectric focusing–double blotting–chemiluminescence detection method presently approved for doping control. Works addressing its advantages and limitations are presented and commented on. The chemical bases of the differential detection are highlighted and some future approaches for detection are also presented. The appearance and detectability of EPO analogues and mimetics susceptible for abuse are also addressed.     

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.     

Doping und Dopinganalytik: Wirkstoffe und Methoden

Since 1997, alterations regarding the anti‐doping rules of sport federations have led to the prohibition of new classes of substances such as plasma volume expanders, anti‐estrogens, aromatase inhibitors and artificial oxygen carriers (e.g. perfluorocarbons, cross‐linked hemoglobins) besides the classical doping agents including stimulants, narcotics, anabolic agents, diuretics and peptide hormones. The determination of doping substances, which has been based mainly on GC‐MS procedures, is more and more performed employing LC‐MS and LC‐MS/MS instruments. For instance, the classes of betablockers, diuretics, corticosteroids and newly proteins such as the cross‐linked hemoglobin 'Hemopure' are effectively detected utilizing LC‐MS(/MS) systems. The urinary identification of erythropoietin (EPO)‐doping is accomplished by means of an assay composed by isoelectric focusing with subsequent visualisation of characteristic EPO bands with monoclonal EPO antibodies (double blotting).     

Cross reactivity between human erythropoietin antibody and horse erythropoietin

Erythropoietin (EPO) is the primary hormone of erythropoiesis. Administration of recombinant human erythropoietin (rhuEPO) to improve racing performance in the horse represents a new form of blood doping, which has been associated with increased mortality. While immunoassay kits have become plentiful, very few commercial hormone assays are made specifically for equine research. There is a strong degree of sequence homology reported for EPO among species, which has allowed antibodies designed for human EPO research to be used to determine EPO concentration in other species. The objective of the present study was to use Western blot analysis to determine whether the antibody to rhuEPO, provided in a commercial radioimmunoassay (RIA) kit, recognizes horse EPO. Western blot analysis of purified rhuEPO and horse plasma was conducted, using the polyclonal goat-antihuman EPO antibody supplied in the Incstar EPO Trac RIA as the primary antibody. Immunoblot analysis revealed a major band at approximately 52 kDa for both rhuEPO and the horse plasma. Our results demonstrate that a human EPO antibody recognizes equine EPO. These findings show that the Incstar EPO Trac RIA hormone assay system can be used to measure equine EPO.     

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis‐stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO‐mimetic peptides is under development from which peginesatide (also referred to as Hematide?), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase‐III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO‐mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL). Copyright © 2011 John Wiley & Sons, Ltd.     

High resolution separation methods for the determination of intact human erythropoiesis stimulating agents. A review

Human erythropoietin (hEPO), a hormone involved in the formation of red blood cells, is a 30 kDa glycoprotein with a high carbohydrate content. The production of recombinant hEPO has made possible its widespread therapeutic use and its banned use in competition sports. Methods to analyze EPO and other erythropoiesis stimulating agents (ESAs) are necessary for the characterization and quality control of these biopharmaceuticals and also for doping control. In this paper, high resolution separation methods, namely high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with special attention to CE-coupled mass spectrometry, are reviewed. The usefulness of these techniques when applied in different modes to separate the glycoprotein isoforms, aggregates or excipients are detailed. In addition, sample preparation methods that have been applied to ESA samples for subsequent determination by HPLC or CE, as well as the potential compatibility of other preparation methods, are discussed. Applications of the HPLC and CE methods regarding regulatory considerations for biopharmaceuticals analysis, with emphasis on biosimilars, and doping control are also included. Finally, limitations of the present methods and their possible solutions are considered.     

Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin

Different profiles were obtained by capillary zone electrophoresis (CZE) of human erythropoietin (EPO) of recombinant and urinary origin. To unambiguously detect doping by EPO, direct methods able to determine the presence of the drug itself in a physiological fluid are required. Since the host cell line used for EPO production influences its glycosylation, the carbohydrate distribution of natural human EPO may be different from that of recombinant EPO. The different content in sialic acid groups between recombinant and endogenous EPO provide a basis for their distinction by CZE.     

Detection of EPO injections using a rapid lateral flow isoform test

Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7–21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today.