Studies on Hyperthyroidism and Wilson’s Disease based on NMR Metabolomics

代谢组学方法是上世纪90年代中期发展起来的一门新学科，它借助高通量、高灵敏度与高精确度的现代分析技术，分析细胞、组织和生物体液中内源性代谢物的整体组成，并通过代谢物复杂的、动态的变化，辨识和解析被研究对象的生理病理状态。本文将核磁共振代谢组学方法应用于两种代谢性疾病的研究中，甲状腺功能亢进是一种代谢性增强的疾病，Wilson病(Wilson’sdisease，WD)是机体内对铜代谢不足导致铜在体内大量积累的疾病。运用核磁共振代谢组学研究方法，分析两种代谢性疾病所导致的代谢改变，寻找了甲亢和Wilson病的特征代谢物。对甲亢疾病的研究中，进一步分析了家族性甲亢的代谢差异。对Wilson病的研究中...The quantitative measurement of the dynamic multi-parametric metabolic response of a living system to pathophysiological stimuli or genetic modification is termed “metabolomics”. The 1H NMR spectroscopy has been shown to be one of the most important analytical techniques used in metabolomics, as it can detect many endogenous metabolites rapidly and reproducibly without tendentiousness or separatio...学位：理学硕士院系专业：材料学院生物材料系_生物医学工程学号：3142008115063

Toxicity Study of VOSO_4 Using NMR-based Metabonomics

通讯作者：陈忠 E-mail: [email protected][中文文摘]采用基于核磁共振(NMR)的代谢组学方法,结合生化指标分析及组织病理学检测,研究了具有类胰岛素活性的硫酸氧钒(VOSO4)对Wistar大鼠的毒性作用.通过不同剂量的VOSO4对Wistar大鼠连续灌胃给药16d,收集大鼠的血清和尿液,并采集样品的1H NMR谱进行多变量数据统计分析来辨识其特征代谢物,然后采用TICL(a web Tool for automatic Interpretation of Compound List)方法建立特征代谢物的代谢网络模型,分析受影响的主要代谢途径及其相互关系.研究结果表明:高剂量组(45mg/kg)和低剂量组(15mg/kg)的特征代谢物含量与对照组存在明显的差异;与对照组相比,高剂量和低剂量组血清中乳酸、肌氨酸酐以及牛磺酸等代谢物的含量增加,尿液中氧化三甲胺(TMAO)、肌酐、牛磺酸和甘氨酸等代谢物的含量增加,并呈现显著的剂量依赖关系;给药组中乙酸和琥珀酸的含量都降低.这些结果说明VOSO4可能影响大鼠体内的糖代谢、脂类代谢及肠道菌群代谢等多个代谢系统,高剂量的VOSO4会导致肝脏毒性和肾脏损伤.[英文文摘]NMR-Based metabonomics combined with clinical biochemical analysis and histopathological examination was applied to investigate the toxicity effects of vanadyl sulfate with insulin-like activity in male Wistar rats. Male Wistar rats were administrated with VOSO4 at doses of 15 and 45 mg/kg body weight by intragastric administration for 16 d. Urine and serum samples were collected and analyzed by 1H NMR experiment. Multivariate analyses were employed to identify the characteristic metabolites for the toxicity effects of vanadyl sulfate.Then the metabolic networks of these characteristic metabolites were built up using TICL (a web Tool for automatic Interpretation of Compound List). The relationship between the characteristic metabolites and the main matebolic pathways perturbed were analyzed and discussed. The differences of metabolic profiles were examined among high-dose (45 mg/kg), low-dose (15 mg/kg) of VOSO4 and control groups. Compared to the control group, increased levels of lactate, creatinine and taurine in serum and increased excretion of trymethylamine-N2-oxide, creatinine, taurine and glycine in urine were found in both high- and low-dose groups which showed an obvious dose-dependent relationship. On the other hand, the concentration of acetate and succinate were decreased in both serum and urine samples of dosed groups. These results indicate that VOSO4 have disturbed the carbohydrate metabolism, lipid metabolism and gut microflora and the high-dosage of VOSO4 may cause liver and kidney injury.卫生部科学研究基金-福建省卫生教育联合攻关计划(No.WKJ2008-2-36);福建省自然科学基金(No.2009J01299)资助项

~1H-NMR Spectroscopy-based Metabonomic Research on Urine of Model Rats of Wilson's Disease

目的:以基于核磁共振(nuClEAr MAgnETIC rESOnAnCE,nMr)的代谢组学方法对WIlSOn病(WIlSOn'S dISEASE,Wd)铜负荷模型大鼠及正常对照组大鼠的尿液进行研究,分析模型大鼠尿液中代谢物的变化,继而从小分子层面探讨铜过量对机体的损伤机制,以更加清楚的认识本病。方法:28只雄性WISTAr大鼠,体重(180±20)g,随机被分为模型组(n=14)和健康对照组(n=14)。采用铜负荷法制作WIlSOn病大鼠模型,以nMr技术对大鼠尿液进行检测。采用MESTrE-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐。对样品进行分段积分,将积分数据归一化后构成数据矩阵,并利用PCA方法对数据矩阵进行统计分析。结果:相对于正常对照组,模型组大鼠尿液醋酸盐(ACETATE)含量有显著升高,柠檬酸盐(CITrATE)、苯乙酰甘氨酸(PAg)、琥珀酸盐(SuCCInATE)、甲胺(METHylAMInE)、肌氨酸+肌氨酸酐(CrEATInE/CrEATInInE)、丙酮酸盐(PyruVATE)、二甲基甘氨酸(dMg)、丙氨酸(AlAnInE)含量有所升高,胆碱(CHOlInE)、牛磺酸(TAurInE)含量有所降低。这些发生改变的代谢物可能是潜在的Wd铜负荷小分子代谢标志物,可为进一步研究Wd的铜过量代谢机制提供参考。Objective :Applying 1H nuclear magnetic resonance(1H-NMR)based metabonomics to study the changes of small molecular metabolites in the urine of the model rats of Wilson's disease.To explore the pathogenesis of Wilson's disease in small molecular aspect.Methods :28 male Wistar rats[weight=(180±20) g] were divided into two groups randomly,the model group(n=14)and the control group(n=14),with the models established by copper-loaded method.Urine of the rats was tested with 1H-NMR technology.The spectra was edited with MestRe-C2.3 and self-programmed software and then principal component analysis(PCA)was applied to differentiate the two groups.Results :Acetate concentration was found to be significantly higher in the urine of the model group;citrate,PAG,succinate,methylamine,creatine/creatinine,pyruvate,DMG,and alanine were higher,and choline and taurine were lower in the urine of the model group.The small molecular metabolites mentioned above may contribute to the discrimination of the two groups,and provide references for further researches on the pathogenesis of WD.安徽省高校自然科学基金重点项目(KJ2012Z228); 安徽中医学院自然科学基金项目(2011ZR008B

--1H-NMR Spectroscopy-based Metabonomic Research on Serum of Model Rats of Wilson's Disease

目的:以基于核磁共振(nMr)的代谢组学方法对WIlSOn病大鼠模型及正常对照组大鼠血清进行研究,分析血清中小分子代谢物的变化,从小分子代谢物层面上探讨WIlSOn病的内在机制,以更加清楚的认识本病。方法:22只雄性WISTAr大鼠,体重(180±20)g,随机被分为模型组(n=11)和健康对照组(n=11),采用铜负荷法制作WIlSOn病大鼠模型,以核磁共振(nMr)技术对大鼠血清进行检测。采用MESTrE-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐。对样品进行分段积分,将积分数据归一化后构成数据矩阵,并利用PCA方法对数据矩阵进行统计分析。结果:相对于正常对照组,模型组大鼠血清甜菜碱(bETAInE)、氧化三甲胺(TAMO)、低密度脂蛋白(ldl)、极低密度脂蛋白(Vldl)、葡萄糖(gluCOSE)含量有显著降低,胆碱(CHOlInE)、胆碱磷酸(PHOSPHOrylCHOlInE)的含量有所降低,乳酸(lACTATE)、谷氨酰胺(gluTAMInE)、糖蛋白(glyCOPrOTEIn)有显著升高,肌氨酸+肌氨酸酐(CrEATInE+CrEATInInE),精氨酸(ArgInInE)有所升高。这些发生改变的代谢物可以作为Wd的小分子代谢标志物,为进一步研究Wd的内在代谢机制提供参考。Objective:Applying 1H nuclear magnetic resonance(1H-NMR)spectroscopy-based metabonomic approach to investigate the changes of small molecular metabolites in the serum from the rats of the model group of Wilson's disease contrasted with those of the control group.Exploring the pathogenesis of Wilson's disease from small molecular aspect.Methods:22 male Wistar rats[weight=(180±20)g]were divided into two groups randomly,the model group(n=11)and the control group(n=11),with the models established with excessive copper method.The serum was tested with 1H-NMR technology.The spectra were edited with MestRe-C2.3 and self-programmed software and then principal component analysis(PCA)was applied to differentiate the two groups.Results:Choline and phosphorylcholine concentrations were found to be lower and TAMO+betaine,LDL,VLDL and glucose were significantly lower in the serum of the model group.While creatinine and arginine concentrations were found to be higher and lactate,glutamine and glycoprotein were significantly higher in the model group.The small molecular metabolites above may contribute to the discrimination,and serve as references for further research on WD pathogenesis.“十一五”国家科技支撑计划分课题重大疑难疾病中医防治研究项目(2006BA104A02

Toxicity Study of VOSO4 Using NMR-based Metabonomics

NMR-Based metabonomics combined with clinical biochemical analysis and histopathological examination was applied to investigate the toxicity effects of vanadyl sulfate with insulin-like activity in male Wistar rats. Male Wistar rats were administrated with VOSO4 at doses of 15 and 45 mg/kg body weight by intragastric administration for 16 d. Urine and serum samples were collected and analyzed by H-1 NMR experiment. Multivariate analyses were employed to identify the characteristic metabolites for the toxicity effects of vanadyl sulfate. Then the metabolic networks of these characteristic metabolites were built up using TICL (a web Tool for automatic Interpretation of Compound List). The relationship between the characteristic metabolites and the main matebolic pathways perturbed were analyzed and discussed. The differences of metabolic profiles were examined among high-dose (45 mg/kg), low-dose (15 mg/kg) of VOSO4 and control groups. Compared to the control group, increased levels of lactate, creatinine and taurine in serum and increased excretion of trymethylamine-N-2-oxide, creatinine, taurine and glycine in urine were found in both high- and low-dose groups which showed an obvious dose-dependent relationship. On the other hand, the concentration of acetate and succinate were decreased in both serum and urine samples of dosed groups. These results indicate that VOSO4 have disturbed the carbohydrate metabolism, lipid metabolism and gut microflora and the high-dosage of VOSO4 may cause liver and kidney injury

Metabonomic Research on Curative Mechanism of Gandouling Treating Model Rats of Wilson's Disease

目的:以基于核磁共振(nuClEAr MAgnETIC rESOnAnCE,nMr)的代谢组学方法对肝豆灵干预WIlSOn病(WIlSOn'S dISEASE,Wd)模型大鼠进行研究,分析肝豆灵干预后,Wd模型大鼠尿液中代谢物的变化,从小分子层面探讨肝豆灵对Wd的治疗作用机制。方法:42只雄性WISTAr大鼠,体重(180±20)g,随机被分为健康对照组(n=14)、模型组(n=14)和肝豆灵组(n=14)。采用铜负荷法制作WIlSOn病大鼠模型,以nMr技术对大鼠尿液进行检测,经MESTrE-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐。对样品进行分段积分,将积分数据归一化后构成数据矩阵,并利用PCA方法对数据矩阵进行统计分析。结果:相对于模型组,肝豆灵组大鼠尿液色氨酸(TryPTOPHAn)、马尿酸盐(HIPPurATE)、苯基丙氨酸(PHEnylAlAnInE)、甘氨酸(glyCInE)、苯乙酰甘氨酸(PAg)含量有显著升高;乳酸(lACTATE)、甜菜碱(TMAO/bETAInE)、甲酸盐(fOrMATE)、二甲基甘氨酸(dMg)含量有所升高;柠檬酸盐(CITrATE)、肌氨酸酐(CrEATInInE)、谷氨酰胺(gluTAMInE)含量显著降低;丙酮酸盐(PyruVATE)甲基胍(METHylguAnIdInE)、丙酮(ACETOnE)、牛磺酸(TAurInE)含量有所降低。这些发生改变的代谢物可为进一步研究肝豆灵干预Wd的作用机制提供参考。Objective:Applying 1H nuclear magnetic resonance(1H-NMR)based metabonomics to study the changes of small molecular metabolites in the urine of model rats of WD treated with Gandouling.To explore the curative mechanism of Gandouling on the model rats in small molecular aspect.Methods:42 male Wistar rats[weight=(180±20)g]were divided into three groups randomly,the model group(n=14),the control group(n=14),and the Gandouling group(n=14),with the models established with copper-loaded method.Urine of the rats was tested with 1H-NMR technology.The spectra were edited with MestRe-C2.3 and self-programmed software and then principal component analysis(PCA)was applied to differentiate the three groups.Results:Compared with those of the model group,tryptophan,hippurate,phenylalanine,glycine and PAG concentration were found to be significantly higher;lactate,TMAO/betaine,formate and DMG were higher;citrate,creatinine and glutamine were significantly lower;pyruvate,methylguanidine,acetone and taurine were lower in the urine of the Gandouling group.The small molecular metabolites mentioned above may contribute to the curative mechanism of Gandouling,and provide references for further researches on the treatment mechanism of WD.安徽省高校自然科学基金重点项目(KJ2012Z228); 安徽中医学院自然科学基金项目(2011ZR008B

NMR-based Metabonomics Study on Serum and Urine of Hyperthyroidism

【中文摘要】应用基于核磁共振(NMR)的代谢组学方法,研究甲状腺功能亢进(简称甲亢)患者和健康人群的血清和尿液,分析甲亢疾病的特征代谢物.实验收集33个甲亢患者和17个健康志愿者的血清样品以及53个甲亢患者和58个健康志愿者的尿液样品,采用多元统计分析方法研究甲亢组和对照组血清和尿液中的内源性代谢差异.结果表明,甲亢组血清中的胆碱、葡萄糖和三甲胺等物质的含量升高,而VLDL,LDL和胆固醇等脂质以及乳酸、糖蛋白和丙氨酸等代谢物的含量下降;甲亢组尿液中的葡萄糖、柠檬酸、牛磺酸以及肌氨酸等代谢物的含量升高,而马尿酸、TMAO、甲酸和琥珀酸等代谢物的含量下降.结果表明,甲亢病不仅影响了糖类、脂类和蛋白质三大物质的代谢,还对能量代谢、肝肠循环和肠道微生物等多个生理系统产生显著影响,并且可能造成肝脏及肾脏等器官的损伤.【Abstract】Nuclear magnetic resonance(NMR) based metabonomics was applied to study the hyperthyroidism by analyzing metabolic profiling of serum and urine. The purpose of this study is to determine an array of characteristic metabolites in serum and urine samples from hyperthyroidism patients and then to interpret these metabolites in possible metabolic pathway. Serum samples from 33 hyperthyroidism patients together with 17 healthy volunteers and urine samples from 53 hyperthyroidism patients with 58 healthy volunteers were collected. Differences in endogenous metabolites were detected on serum and urine samples from the hyperthyroidism group and control group using multivariate statistical analysis. The results show that the hyperthyroidism group has elevated levels of choline, glucose and declined levels of VLDL, LDL, cholesterol, lactate, glycoprotein and alanine in serum samples relative to the healthy group. In urine samples, the hyperthyroidism group show increased levels of glucose, citrate, taurine and creatinine as well as decreased levels of hippurate, timethyl-amine-N-oxide, formate and succinate. These results indicate that hyperthyroidism have not only disturbed the carbohydrate metabolism, lipid metabolism and protein metabolism but also influenced energy metabolism, hepatoenteral circulation and gut microflora and caused liver and kidney injury

NMR-based Metabolomic Study on Serum and Urine of Familial Hyperthyroidism

采用基于核磁共振(nMr)的代谢组学方法,研究了家族性甲亢患者、非家族性甲亢患者和健康志愿者人群的血清和尿液的代谢差异.结果表明,家族性甲亢组血清样品中的乙酰乙酸、柠檬酸及牛磺酸等代谢物的浓度显著升高,超低密度脂蛋白、低密度脂蛋白、乳酸及甘氨酸等代谢物的浓度显著降低;尿样中的肌酐和葡萄糖的浓度显著升高,琥珀酸、苯乙酰甘氨酸及马尿酸等代谢物的浓度显著降低.以上结果表明家族性甲亢患者可能存在糖和脂类代谢的紊乱、肝脏和肾脏功能的损伤以及肠道环境的破坏等.此外,家族性甲亢组血清样品中的乙酰乙酸、葡萄糖、柠檬酸及肌酐等代谢物的浓度显著升高,表明家族性甲亢人群的肾脏功能受损更为严重,由此可区分家族性与非家族性甲亢的nMr代谢轮廓.Nuclear magnetic resonance(NMR) based on metabonomics was applied to study of the differences in endogenous metabolites on serum and urine samples from the familial hyperthyroidism group,non-familial hyperthyroidism group and control group using multivariate statistical analysis in this paper.The experimental results show that the familial hyperthyroidism group has significant difference with the non-familial hyperthyroidism group and the control group.The familial hyperthyroidism group has elevated levels of acetoacetate,citrate and taurine,and has declined levels of very low density lipoprotein,low density lipoprotein,lactate and glycine in serum samples relative to the control group.In urine samples,the familial hyperthyroidism group show increased levels of glucose and creatinine as well as decreased levels of succinate,phenacetylglycine and hippurate.These results would lead to the disorder of carbohydrate and lipid metabolism,the injury of liver and kidney,cellular toxicity and the destruction of the intestinal environment.Furthermore,relative to the non-familial hyperthyroidism group,the familial hyperthyroidism has more significantly increased levels of the acetoacetate,glucose,citrate and creatinine in the serum.Therefore,the injury of kidney function and cell is more serious in familial hyperthyroidism group.国家自然科学基金(批准号:81201143;81371639); 中央高校基本科研业务费(批准号:2013121007)资