高血压病的代谢组学研究
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摘要
代谢组学又称代谢物组学,是研究机体代谢产物谱变化的一种新的系统方法。作为一种“组学”科学,它借助高通量、高灵敏度与高精确度的现代分析技术,来分析细胞、组织和体液如血液或尿液中内源性代谢物整体组成,通过研究代谢物的动态变化来辩识和解析被研究对象的生理病理状态。作为崭新的方法学,代谢组学已成为国际上疾病与健康研究的一个重要热点。因此采用代谢组学方法来研究高血压病,对于高血压病的早期诊断、预防和药物干预都具有重要的指导意义。
本论文采用HPLC法和ICP-MS法作为代谢组学研究的检测手段,以高血压患者和正常人的血浆和尿液为研究对象,测定其中各类代谢产物的含量。高血压患者和正常人血浆和尿液的代谢产物图谱,目视难以区分两者的差异。采用模式识别方法,能够成功区分高血压患者和正常人血浆和尿液样本。说明通过血浆和尿液中的代谢产物分析能够有效辨识高血压状态。本研究采用代谢组学方法研究高血压病,为高血压病的早期诊断、预防和致病机理研究提供了理论依据。
1.反相高效液相色谱法测定尿液中非蛋白氮代谢产物
建立了RP-HPLC法测定尿液中5种非蛋白氮代谢产物(尿素、肌酸、肌酐、尿酸和马尿酸)的含量。尿样经离心和稀释后,C_(18)柱分离,以乙腈-醋酸铵缓冲液(20 mmol·L~(-1),pH6.8)为流动相,梯度洗脱,检测波长220 nm。肌酸、肌酐、尿酸和马尿酸浓度在2～100mg·mL~(-1)内,尿素浓度在100～1000 mg·mL~(-1)内线性关系良好(γ＞0.99),方法平均回收率为97.6%～105%,RSD为0.79～4.2%(n=6)。该方法操作简便、快速,灵敏度高,重复性好,适用于尿液中非蛋白氮代谢产物的快速分析。
2.柱前衍生高效液相色谱法测定血浆和尿液中氨基酸
建立了RP-HPLC法测定血浆和尿液中22种氨基酸的含量。以丹磺酰氯为柱前衍生化试剂,血浆样品和尿样经沉淀蛋白处理后,C_(18)柱分离,以醋酸钠-甲醇-四氢呋喃为流动相,梯度洗脱,荧光检测。氨基酸浓度在2～400μmol·L~(-1)内线性关系良好(γ＞0.99),方法平均回收率为82.0%～109%,RSD为0.98～7.3%(n=6)。该方法精密度和重复性好,适用于血浆和尿液中氨基酸的测定。
3.电感耦合等离子体质谱法测定尿液中微量元素
建立了ICP-MS法测定尿液中16种微量元素的含量。尿样经离心和稀释后进样分析。仪器测试条件为:射频功率1570 W;载气流速1.2 L·min~(-1);等离子体气流速14.9L·min~(-1);雾化室温度2℃。Al、V、Cr、Mn、Co、Ni、Cu、Zn、As、Se、Mo、Cd、Ba和Pb浓度在0.1～100μg·L~(-1)内,Mg和Fe浓度在10～10000μg·L~(-1)内线性关系良好(γ＞0.999),检出限为2～143 ng·L~(-1),仪器精密度为0.25%～3.7%(n=10),方法精密度为0.79%～4.9%(n=6)。该方法简便、快速,精密度好,灵敏度高,适用于尿液中微量元素的高通量、快速分析。
4.数据处理及结果分析
采用SPSS13.0统计软件,对高血压患者和正常人体内的氨基酸和微量元素含量分别进行主成份分析和判别分析,结果表明:高血压组和正常对照组体内氨基酸和微量元素的代谢模式存在显著性差异。主成份分析可以将高血压患者和正常人的血浆和尿液样本明显区分开,分类判别准确率大于98%。高血压患者体内氨基酸代谢异常,这可能与其饮食结构或体内氨基酸失衡有关;而高血压患者体内微量元素代谢紊乱,除了与饮食结构和遗传因素有关外,环境的影响可能也是一个重要的原因,进一步的研究仍在进行中。
Metabonomics or metabolomics is the quantitative measurement of the multiparametric time-related metabolic responses of a complex system to a pathophysiological intervention or genetic modification.Metabolomics apply useful modeling tools for the classification and prediction of physiological and pathological states from metabolite profiles of biofluids such as plasma and urine.Thus metabonomics seeks to assess the global system level homeostatic and pathological responses to interventions or stressors.The main characteristics of metabonomics are the high-throughput experiment and calculation.As a new methodology, metabonomics has become an important hotspot for the research of disease and healthy in the world now.Therefore,to research hypertension with metabonomic method is significant for the early-stage diagnose and prevention of disease and drug intervention.
In the present study,we have developed HPLC and ICP-MS approaches as determination means of metabolomics,to differentiate healthy persons and hypertension patients.Plasma and urine metabolite content from healthy persons and hypertension patients were acquired using HPLC and ICP-MS methods.It was unable to distinguish plasma and urine metabolite chromatography from healthy persons and hypertension patients.Using pattern recognition,it was capable of distinguishing normal blood pressure plasma and urine samples from hypertension patients.It was showed that the analysis of metabolite in plasma and urine can recognize hypertension condition.The present study was a successful research of hypertension using metabolomics approach.The study provided theory for the early-stage diagnose and prevention of disease and pathopoiesis mechanism.
1.Determination of nonprotein-nitrogen metabolites in urine by RP-HPLC method
To establish a RP-HPLC method for determinating 5 kinds of nonprotein-nitrogen metabolites in urine(urea,creatine,creatinine,uric acid and hippuric acid).The urine samples were centrifugated and diluted prior to analysis.The five compounds were successfully separated on a C_(18)column using a mobile phase of acetonitrile-ammonium acetate (20 mmol·L~(-1),pH 6.8)with gradient elution.The detection wavelength was selected at 220 nm.The method was linear over the concentration range of 2 to 100 mg·mL~(-1)for creatine,uric acid,creatinine and hippuric acid,and 100 to 1000 mg·mL~(-1)for urea(γ＞0.99).The mean recovery was between 97.6%and 105%.The RSD was between 0.79%and 4.2%(n=6).The method was simple,rapid,sensitive and accurate,and was suitable for rapid analysis of nonprotein-nitrogen metabolites in urine.
2.Determination of amino acids in plasma and urine by pre-column derivatization HPLC method
A RP-HPLC method for determination of 22 amino acids in plasma and urine was established.Dansyl chloride was used as pre-column derivatization agent.The plasma and urine samples were precipitated protein prior to analysis.The 22 amino acid derivatives were successfully separated on a C_(18)column using a mobile phase of sodium acetate-methanoltetrahydrofuran with gradient elution.The fluorescence detection was used for determination. The assay was linear over the concentration range of 2 to 400μmol·L~(-1)for all amino acids (γ＞0.99).The mean recoveries were between 82.0%and 109%.The RSDs were between 0.98%and 7.3%(n=6).It indicated that the method has good precision and repeatability.The method was suitable to determinate amino acids in plasma and urine.
3.Determination of trace element in urine by inductive coupled plasma mass spectrometry method
The paper presented a method for the fast,simple and reliable routine determination of 16 trace elements in urine by ICP-MS.The urine samples were centrifugated and diluted prior to analysis.ICP-MS operating conditions:power 1570 W;outer gas flow 1.2 L·min~(-1);plasma gas flow 14.9 L·min~(-1);spray chamber temperature 2℃.The assay was linear over the concentration range of 0.1 to 100μg·L~(-1)for Al、V、Cr、Mn、Co、Ni、Cu、Zn、As、Se、Mo、Cd、Ba and Pb,and 10 to 10000μg·L~(-1)for Mg and Fe(γ＞0.999).The limit of determination was between 2 to 143 ng·L~(-1).The precision of instrument was between 0.25% and 3.7%(n=10).The precision of method was between 0.79%and 4.9%(n=6).The method was simple,rapid and sensitive,and suitable for determination of trace element in urine.
4.Data processing and result analysis
Principal component analysis and discriminant analysis with SPSS13.0 statistics software,were used to process amino acid and trace element concentration from healthy people and hypertension patients.The result indicated that there was significance differentiation in amino acids and trace element metabolism mode between healthy people and hypertension patients.Using principal component analysis,it was capable of distinguishing normal blood pressure plasma and urine samples from hypertension patients. The classification accuracy was more than 98%.There was metabolism abnormality of amino acids in hypertension patients.The diet structure may affect the disorder.Disequilibrium of amino acids may be another reason.There was metabolism abnormality of trace element in hypertension patients.Besides diet structure and heredity factor,environment factor may be another important reason.Further study is in progress.
本论文采用HPLC法和ICP-MS法作为代谢组学研究的检测手段,以高血压患者和正常人的血浆和尿液为研究对象,测定其中各类代谢产物的含量。高血压患者和正常人血浆和尿液的代谢产物图谱,目视难以区分两者的差异。采用模式识别方法,能够成功区分高血压患者和正常人血浆和尿液样本。说明通过血浆和尿液中的代谢产物分析能够有效辨识高血压状态。本研究采用代谢组学方法研究高血压病,为高血压病的早期诊断、预防和致病机理研究提供了理论依据。
1.反相高效液相色谱法测定尿液中非蛋白氮代谢产物
建立了RP-HPLC法测定尿液中5种非蛋白氮代谢产物(尿素、肌酸、肌酐、尿酸和马尿酸)的含量。尿样经离心和稀释后,C_(18)柱分离,以乙腈-醋酸铵缓冲液(20 mmol·L~(-1),pH6.8)为流动相,梯度洗脱,检测波长220 nm。肌酸、肌酐、尿酸和马尿酸浓度在2～100mg·mL~(-1)内,尿素浓度在100～1000 mg·mL~(-1)内线性关系良好(γ＞0.99),方法平均回收率为97.6%～105%,RSD为0.79～4.2%(n=6)。该方法操作简便、快速,灵敏度高,重复性好,适用于尿液中非蛋白氮代谢产物的快速分析。
2.柱前衍生高效液相色谱法测定血浆和尿液中氨基酸
建立了RP-HPLC法测定血浆和尿液中22种氨基酸的含量。以丹磺酰氯为柱前衍生化试剂,血浆样品和尿样经沉淀蛋白处理后,C_(18)柱分离,以醋酸钠-甲醇-四氢呋喃为流动相,梯度洗脱,荧光检测。氨基酸浓度在2～400μmol·L~(-1)内线性关系良好(γ＞0.99),方法平均回收率为82.0%～109%,RSD为0.98～7.3%(n=6)。该方法精密度和重复性好,适用于血浆和尿液中氨基酸的测定。
3.电感耦合等离子体质谱法测定尿液中微量元素
建立了ICP-MS法测定尿液中16种微量元素的含量。尿样经离心和稀释后进样分析。仪器测试条件为:射频功率1570 W;载气流速1.2 L·min~(-1);等离子体气流速14.9L·min~(-1);雾化室温度2℃。Al、V、Cr、Mn、Co、Ni、Cu、Zn、As、Se、Mo、Cd、Ba和Pb浓度在0.1～100μg·L~(-1)内,Mg和Fe浓度在10～10000μg·L~(-1)内线性关系良好(γ＞0.999),检出限为2～143 ng·L~(-1),仪器精密度为0.25%～3.7%(n=10),方法精密度为0.79%～4.9%(n=6)。该方法简便、快速,精密度好,灵敏度高,适用于尿液中微量元素的高通量、快速分析。
4.数据处理及结果分析
采用SPSS13.0统计软件,对高血压患者和正常人体内的氨基酸和微量元素含量分别进行主成份分析和判别分析,结果表明:高血压组和正常对照组体内氨基酸和微量元素的代谢模式存在显著性差异。主成份分析可以将高血压患者和正常人的血浆和尿液样本明显区分开,分类判别准确率大于98%。高血压患者体内氨基酸代谢异常,这可能与其饮食结构或体内氨基酸失衡有关;而高血压患者体内微量元素代谢紊乱,除了与饮食结构和遗传因素有关外,环境的影响可能也是一个重要的原因,进一步的研究仍在进行中。
Metabonomics or metabolomics is the quantitative measurement of the multiparametric time-related metabolic responses of a complex system to a pathophysiological intervention or genetic modification.Metabolomics apply useful modeling tools for the classification and prediction of physiological and pathological states from metabolite profiles of biofluids such as plasma and urine.Thus metabonomics seeks to assess the global system level homeostatic and pathological responses to interventions or stressors.The main characteristics of metabonomics are the high-throughput experiment and calculation.As a new methodology, metabonomics has become an important hotspot for the research of disease and healthy in the world now.Therefore,to research hypertension with metabonomic method is significant for the early-stage diagnose and prevention of disease and drug intervention.
In the present study,we have developed HPLC and ICP-MS approaches as determination means of metabolomics,to differentiate healthy persons and hypertension patients.Plasma and urine metabolite content from healthy persons and hypertension patients were acquired using HPLC and ICP-MS methods.It was unable to distinguish plasma and urine metabolite chromatography from healthy persons and hypertension patients.Using pattern recognition,it was capable of distinguishing normal blood pressure plasma and urine samples from hypertension patients.It was showed that the analysis of metabolite in plasma and urine can recognize hypertension condition.The present study was a successful research of hypertension using metabolomics approach.The study provided theory for the early-stage diagnose and prevention of disease and pathopoiesis mechanism.
1.Determination of nonprotein-nitrogen metabolites in urine by RP-HPLC method
To establish a RP-HPLC method for determinating 5 kinds of nonprotein-nitrogen metabolites in urine(urea,creatine,creatinine,uric acid and hippuric acid).The urine samples were centrifugated and diluted prior to analysis.The five compounds were successfully separated on a C_(18)column using a mobile phase of acetonitrile-ammonium acetate (20 mmol·L~(-1),pH 6.8)with gradient elution.The detection wavelength was selected at 220 nm.The method was linear over the concentration range of 2 to 100 mg·mL~(-1)for creatine,uric acid,creatinine and hippuric acid,and 100 to 1000 mg·mL~(-1)for urea(γ＞0.99).The mean recovery was between 97.6%and 105%.The RSD was between 0.79%and 4.2%(n=6).The method was simple,rapid,sensitive and accurate,and was suitable for rapid analysis of nonprotein-nitrogen metabolites in urine.
2.Determination of amino acids in plasma and urine by pre-column derivatization HPLC method
A RP-HPLC method for determination of 22 amino acids in plasma and urine was established.Dansyl chloride was used as pre-column derivatization agent.The plasma and urine samples were precipitated protein prior to analysis.The 22 amino acid derivatives were successfully separated on a C_(18)column using a mobile phase of sodium acetate-methanoltetrahydrofuran with gradient elution.The fluorescence detection was used for determination. The assay was linear over the concentration range of 2 to 400μmol·L~(-1)for all amino acids (γ＞0.99).The mean recoveries were between 82.0%and 109%.The RSDs were between 0.98%and 7.3%(n=6).It indicated that the method has good precision and repeatability.The method was suitable to determinate amino acids in plasma and urine.
3.Determination of trace element in urine by inductive coupled plasma mass spectrometry method
The paper presented a method for the fast,simple and reliable routine determination of 16 trace elements in urine by ICP-MS.The urine samples were centrifugated and diluted prior to analysis.ICP-MS operating conditions:power 1570 W;outer gas flow 1.2 L·min~(-1);plasma gas flow 14.9 L·min~(-1);spray chamber temperature 2℃.The assay was linear over the concentration range of 0.1 to 100μg·L~(-1)for Al、V、Cr、Mn、Co、Ni、Cu、Zn、As、Se、Mo、Cd、Ba and Pb,and 10 to 10000μg·L~(-1)for Mg and Fe(γ＞0.999).The limit of determination was between 2 to 143 ng·L~(-1).The precision of instrument was between 0.25% and 3.7%(n=10).The precision of method was between 0.79%and 4.9%(n=6).The method was simple,rapid and sensitive,and suitable for determination of trace element in urine.
4.Data processing and result analysis
Principal component analysis and discriminant analysis with SPSS13.0 statistics software,were used to process amino acid and trace element concentration from healthy people and hypertension patients.The result indicated that there was significance differentiation in amino acids and trace element metabolism mode between healthy people and hypertension patients.Using principal component analysis,it was capable of distinguishing normal blood pressure plasma and urine samples from hypertension patients. The classification accuracy was more than 98%.There was metabolism abnormality of amino acids in hypertension patients.The diet structure may affect the disorder.Disequilibrium of amino acids may be another reason.There was metabolism abnormality of trace element in hypertension patients.Besides diet structure and heredity factor,environment factor may be another important reason.Further study is in progress.
引文
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