肌酐、磷脂代谢物检测新方法

发布时间：2018-06-16 23:55

本文选题：代谢组学 + 肌酐　；参考：《新疆大学》2016年硕士论文

【摘要】：代谢组学是研究生物体系受外部刺激或扰动后所产生的内源性代谢物整体及其变化规律的科学。血浆和尿液作为代谢组学的研究中最常用两种体液样本,对其所含的代表性代谢物进行快速准确分离与分析具有十分重要的理论价值和应用前景。本研究以人体体液中的两种(类)代表性代谢物肌酐和磷脂类化合物为研究对象,通过建立新的检测方法,实现对它们的高选择、高准确性的分离与分析。此外,建立了基于液质联用技术的人体尿液代谢轮廓分析方法,为代谢组学研究提供新的技术手段。具体研究内容如下:(1)基于LbL技术将PEI/PTA多层膜构建于氧化铟导电玻璃片上作为化学修饰电极,采用循环伏安法,在铜离子辅助下测定不同浓度标准溶液中的肌酐含量,标准曲线的线性范围为0.125~62.5μM,检出限达到0.06μM,采用标准加入法得到平均回收率分别为106.7%、96.49%和99.86%(n=3)。将该电极应用于人体尿液中的肌酐含量的检测,然后用HPLC法对该测定结果进行验证,结果无显著性差异,说明该电极可准确测定体液中的肌酐,具有良好的临床应用前景。(2)基于化学共沉淀反应原理,采用“一锅法”合成了乙二胺四乙酸(EDTA)功能化的磁性纳米粒子(EDTA@Fe3O4)。将Zr(IV)螯合于磁性纳米颗粒表面制得Zr(IV)-IMAC,并通过TEM、EDS等方法对其进行表征。将Zr(IV)-IMAC用于富集四种磷脂标准品,首先建立了HPLC-ELSD同时测定的方法并对富集过程中的洗脱液进行了优化,发现在上清液和洗涤液中均未检出,仅在洗脱液中可以检测出四种磷脂标准品,回收率均大于94.23%,说明Zr(IV)-IMAC对磷脂具有很好的选择性。将之用于实际血浆样品中磷脂的富集,并用UPLC-QTOF进行检测,经HMDB数据库检索,初步鉴定出可能的47种磷脂类化合物。(3)为了探究不同民族(汉族和维吾尔族)因素对人体尿液代谢轮廓的影响,共收集健康志愿者尿液样本128例,维吾尔族和汉族各64例。优化了色谱质谱条件,建立了基于UPLC-QTOF平台的代谢轮廓分析方法,并完成20例样本分析,其中包括汉族和维吾尔族各10例。
[Abstract]:Metabonomics is the science of studying the whole and changing law of endogenous metabolites produced by external stimulation or disturbance of biological system. Plasma and urine are the two most commonly used body fluid samples in the study of metabolomics. The rapid and accurate separation and analysis of representative metabolites contained in plasma and urine have very important theoretical value and application prospect. In this study, two representative metabolites (creatinine and phospholipid compounds) in human body fluid were studied. A new detection method was established to separate and analyze them with high selectivity and accuracy. In addition, a method of urine metabolic profile analysis based on liquid-mass spectrometry was established, which provides a new technique for the study of metabolomics. The specific research contents are as follows: (1) based on LBL technique, PEI / PTA multilayer film was constructed on indium oxide conductive glass as chemically modified electrode, and the creatinine content in different concentration standard solution was determined by cyclic voltammetry under the assistance of copper ion. The linear range of the standard curve was 0.125 渭 m and the detection limit was 0.06 渭 M. the average recoveries of the standard addition method were 96.49% and 99.86%, respectively. The electrode was applied to the determination of creatinine in human urine, and the results were verified by HPLC. The results showed that the electrode could accurately determine creatinine in body fluid. Based on the principle of chemical coprecipitation, EDTA-EDTA-functionalized magnetic nanoparticles (EDTA-Fe3O4) were synthesized by one-pot method. ZrOIV-IMAC was prepared by chelating ZrOIV onto the surface of magnetic nanoparticles and characterized by TEM-EDS and other methods. The method of simultaneous determination of HPLC-ELSD was established for the enrichment of four phospholipid standard products by ZrAI-IV-IMAC. The eluate in the enrichment process was optimized. It was found that the eluate was not detected in the supernatant and washing liquid. Four kinds of phospholipid standard compounds can be detected only in elution solution. The recoveries of phospholipids are all over 94.230.The results show that ZrAI-IV-IMAC has good selectivity for phospholipids. It was used for the enrichment of phospholipid in actual plasma samples, and was detected by UPLC-QTOF, and searched by HMDB database. In order to explore the influence of different nationalities (Han and Uygur) on the metabolic profile of human urine, 128 urine samples were collected from healthy volunteers, 64 from Uygur and 64 from Han nationality. Based on UPLC-QTOF platform, the metabolic profile analysis method was established, and 20 samples were analyzed, including 10 Han and 10 Uygur.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.1

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