基于GC/TOF-MS小鼠血浆代谢组学技术方法的建立及其应用
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摘要
代谢组学是一门新兴的组学技术,是研究生命体受外部刺激所产生的所有代谢产物的变化,是基因组学和蛋白组学的延伸,反映的是已经发生了的生物学事件,代谢组学研究,除了研究药物本身的代谢变化外,主要是研究药物引起的内源性代谢物的变化,更直接反映了体内生化过程和微环境的改变,可全面了解代谢物质在疾病发生、发展过程中的变化规律,为疾病的预防和诊断提供新的思路。
目前,代谢组学已广泛应用在药物毒性试验、功能基因组、临床诊断、临床治疗、疗效评估、营养学和分子流行病学等领域,但仍处在初步研究阶段尤其是在分析方法方面,怎样借助相关的分析手段建立一种合理的分析方法仍是一个急待解决的难题。
本论文构建了基于GC/TOF-MS小鼠血浆代谢组学分析方法,以及合适的数据处理、模式识别和统计学分析方法,并应用于D-GalN/LPS诱导的暴发性肝衰竭小鼠模型血浆代谢物谱分析,寻找相关代谢标志物,为暴发性肝衰竭的早期诊断奠定基础。
一、基于GC/TOF-MS的小鼠血浆代谢组学分析方法的建立
本实验在相关文献基础上,进一步考察不同有机溶剂的配比对内源性代谢产物提取效率的影响,并分别将水相、氯仿相以及水和氯仿混合相吹干衍生化进行GC/TOF-MS分析,利用相关统计学方法,研究了不同有机相分层后,各层对代谢物提取的影响,实验结果表明：同时加入氯仿、水和甲醇分层后,将水和甲醇混合相吹干衍生化,并通过GC/TOF-MS分析,得到总离子流色谱图中,无论是峰的个数还是峰的强度都优于其它方法。
将总离子流色谱图中每个色谱峰,借助AMDIS软件去卷积,通过NIST05标准谱库进行检索。结果显示,共监测到总代谢物峰269个,通过与标准谱库比对,55个谱峰相似度在700以上,认为是比较可靠的代谢物。并在检测到的55个代谢产物中任意选取18种,以核糖醇作为内标,考察基于GC/TOF-MS技术的稳定性,实验结果表明,系统稳定性、制样稳定性以及4℃存放一周的稳定性,其变异系数均在15以内,证明此方法稳定可行。在此方法基础上,利用氨基酸和单糖的标准品对相关峰进一步指认,建立了基于GC/TOF-MS方法的大鼠血浆内源性代谢物谱库,总共包含38个代谢产物,为以后血浆中GC/TOF-MS代谢物谱图的鉴定提供方便,提高工作效率。
二、利用已建方法进行D-氨基半乳糖/内毒素诱导的暴发性肝衰竭小鼠模型代谢组学分析
本实验将12只雄性BALB/c小鼠,随机分为D-Gal/LPS诱导的FHF组(6只)和对照组(6只)。根据质谱数据,监测到最多267个峰,其中52个被证实为糖、氨基酸、脂肪酸和有机酸等内源性代谢物。52个代谢物中,49个被识别而其他3个尽管未被识别但在两组之间有明显差异。这些物质广泛参与了机体的物质代谢和能量代谢的生化过程。
根据GC/TOF-MS色谱,D-Gal/LPS处理后血浆氨基酸水平发生了明显变化。与对照组比较,所识别的16种氨基酸在FHF组含量均有升高,除色氨酸的升高无统计学意义外,其余15种氨基酸(丙氨酸、缬氨酸、异亮氨酸、甘氨酸、丝氨酸、苏氨酸、脯氨酸、亮氨酸、天冬氨酸、苯丙氨酸、谷氨酰胺、谷氨酸、鸟氨酸、赖氨酸和酪氨酸)均明显升高,血浆中总氨基酸和生糖氨基酸的含量也明显升高。另外,FHF组BCAA/AAA值明显降低。
与对照组比较,非氨基有机酸中FHF组p-羟丁酸(HB)、延胡索酸、苹果酸、2,3,4-三羟基丁酸、尿酸等明显升高,而烯醇式丙酮酸(EP)、琥珀酸、十四烷酸明显降低。碳水化合物中除半乳糖和D-阿卓糖明显降低外,包括葡萄糖在内的其他化合物均无明显变化。另外,FHF组血浆磷酸、γ-氨基丁酸(GABA)、5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA)水平升高,而尿酸、软脂酸、硬脂酸含量均无异常变化。
依据血浆代谢物水平变化及D-Gal/LPS损伤肝脏的机制,所涉及代谢途径包括糖异生、糖酵解、酮体产生、三羧酸循环和尿素循环等。结果显示,与对照组比较,FHF组糖异生和糖酵解途径无明显变化,但酮体产生、三羧酸循环和尿素循环受到抑制。尽管几乎所有氨基酸水平升高,但只有少数如天冬氨酸、苯丙氨酸和酪氨酸促进了糖异生。
由于生化途径互相联系、高度调节、错综复杂,而且平均化处理的过程中会丢失信息,代谢组学的数据处理较为困难。诸如主成分分析等多变量统计学分析方法可以通过将大量的数据信息集中于一个特定数据组来减少数据复杂性。通过对两组小鼠血浆样本的GC/TOF-MS数据进行主成分分析,可以看出FHF组和对照组存在聚类特征且两组间存在明显的类别差异,使用SIMCA-P软件发现,代谢产物中5-HIAA、葡萄糖、HB和磷酸是对两个实验组的差异贡献值最大。
总之,本论文构建了基于GC/TOF-MS的小鼠血浆样本代谢组学分析方法,通过方法学考察表明该方法具有较好的实验精密度、线性和稳定性。并将该分析方法应用于D-氨基半乳糖／内毒素诱导的暴发性肝衰竭小鼠模型。通过与对照组比较,在D-Gal/LPS诱导的FHF小鼠模型,许多血浆代谢物水平发生了明显变化。而且,FHF早期的代谢变化中酮体生成、TCA和尿素循环受到抑制,但糖异生和糖酵解途径无明显变化。PCA数据分析显示5-HIAA、葡萄糖、HB和磷酸水平的组合是暴发性肝衰竭的潜在可靠标志物。以上结果说明这种方法可作为有力工具用以了解病理生理条件下代谢物水平的变化和进行相关代谢组学研究、寻找代谢标志物,并可用于暴发性肝衰竭的早期诊断。
Metabonomics has been increasingly highlighted in "omics" technology, which is to study the systematic changes of all the metabolites in response to external stimuli. Metabonomics is the extension of genomics and proteomics, reflecting the biological event that has occurred. Metabonomics research, in addition to the changes of drug metabolism themselves, centers on the drug-induced changes of endogenous metabolites, which can shed light on the process of diseases comprehensively and offer new ideas about disease prevention and diagnosis.
Currently, metabonomics has been widely used in toxicology, genomics, clinical diagnosis, clinical treatment, assessment of treatment, nutrition and molecular epidemiology, most of which are still in the preliminary research stage, especially in the analytical method. How to establish a rational method is still an urgent problem to be solved.
A metabonomic method, based on GC/TOF-MS of endogenous metabolites in small rat serum, as well as on the appropriate data processing, pattern recognition and statistical analysis methods, has been successfully applied to D-GalN/LPS-induced mouse model of fulminant hepatic failure(FHF) and facilitate the search for metabolic markers that, if linked to substance pathways, may also be used for early prognosis of FHF.
Firstly, a metabonomic method was established based on GC/TOF-MS of endogenous metabolites in plasma of mice.
plasma metabolite extraction conditions based on relevant literatures were developed, The extraction efficiency of different organic solvents, singly and in combination, was investigated to optimize the extraction condition of endogenous metabolites.
The result of the method,500μL of methanol,20μL of ribitol,450μL of pure water and 250μL of chloroform were added in Plasma samples(100μL) simultaneously, showed that its number and intensity of peak surpassed those of other methods By relevant statistical analysis.
On the basis of the mass spectral database(NIST05) and AMDIS software,269 peaks were detected in plasma samples, the similarity of 48 of which was above 7000 and confirmed to be endogenous metabolites in the TIC detected by GC/TOF-MS.
Eighteen endogenous metabolites were selected to investigate the system stability based on GC/TOF-MS technology at random from forty-eight metabolites.The result indicated most of the metabolites demonstrated good reproducibility, with intra-day, inter-day and stored in 4℃precision values below 15%. The relevant peaks were confirmed by standard compounds of monosaccharides and amino acids. The metabolites libraries of plasma were thus established, including 38 metabolites in the plasma library. These database can facilitate increasing the speed and accuracy of the peaks'identification in TIC.
Secondly, Metabonomic Analysis for D-GalN/LPS-induced mouse model of Fulminant Hepatic Failure(FHF).
12 male BALB/c mice were housed in a standard animal laboratory with a 12 h light-dark cycle and were provided with water and standard mouse chow ad libitum. The animals were randomly divided into GalN/LPS-induced FHF(n=6) and control(n=6)groups.
On the basis of the mass spectral database,267 peaks were detected,52 of which were confirmed to be endogenous metabolites, such as sugars, amino acids, fatty acids, and organic acids, among other compounds. Of the 52 metabolites,48 were identified, whereas the others were unidentified metabolites in which significant differences were noted between the FHF group and the controls. These substances have been implicated in multiple biochemical processes, including energy and substance metabolism.
Marked changes in plasma amino acids were observed on GC/TOF-MS chromatograms following GalN/LPS treatment. Though 16 amino acids were identified in total, amino acid concentrations were generally elevated in the FHF group, with significant differences in the levels of 15 amino acids(alanine,valine, isoleucine, glycine, serine, threonine, proline, leucine,aspartate, phenylalanine, glutamine, glutamate, ornithine,lysine, and tyrosine), with the exception of tryptophan, in which the increase was not statistically significant. The concentrations of total amino acids and gluconeogenic amino acids also increased. Furthermore, the ratio of BCAA to AAA (valine+leucine+isoleucine)/(tyrosine+phenylalanine+tryptophan) was significantly reduced in the FHF group.
Compared with the control group,β-hydroxybutyrate(HB), fumarate, aminomalonic acid, malate and uric acid were elevated in the treatment group. Interestingly, levels of enolpyruvate(EP), succinate, and tetradecanoic acid decreased significantly among the nonamino-organic acids identified in FHF mice. Although there was a noted decrease in galactose and D-altrose in these mice, no significant differences in other carbohydrates, including glucose, were noted. Elevated phosphate,γ-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT), and 5-hydroxyindol-eaceti acid(5-HIAA)levels were also observed after GalN/LPS treatment. Levels of urea, palmic acid and stearic acid were similar in both groups.
based on changes in the levels of intermediates in the FHF mice and the mechanisms underlying GalN/LPS damage to the liver, the pathways included gluconeogenesis, glycolysis, production of ketone bodies, the tricarboxylic acid(TCA) and urea cycles, among others. Whereas no significant differences between the fasted control and treated mice were noted in the gluconeogenic and glycolytic pathways, the production of ketone bodies, the TCA cycle, and the urea cycle were inhibited in the FHF mice. Although the levels of almost all amino acids were elevated in these mice, only a minority(e.g., aspartate, phenylalanine, and tyrosine) contributed to gluconeogenesis.
Interpretation of the metabolite level data is difficult, not only because biochemical pathways are linked and highly regulated but also because information is lost in the process of averaging. Analysis methods, such as PCA, reduce data complexity by focusing on the information content of a given data set, while maintaining most of the information in a few dimensions. Analysis with SIMCA-P and PCA softwares indicated distinct clustering was observed between the FHF group and the control group.5-HIAA, glucose, HB, and phosphate are the parameters with the highest weights, and thus they had the greatest impact on each of the principal components in each experimental group.
In all, based on GC/TOF/MS metabonomic method was establishped and applied for GalN/LPS-induced FHF. By the analysis of the metabolic profiling of plasma, significant differences in the plasma levels of many metabolites were noted, compared with those of the controls in the model of GalN/LPS-induced FHF. Furthermore, the earliest metabolic perturbations detected included the inhibition of ketogenesis and the TCA and urea cycles, with no significant changes in the gluconeogenic and glycolic pathways. PCA data analysis suggests that a combination of 5-HIAA, glucose, HB, and phosphate concentrations in the plasma is a potential marker for FHF. The above results demonstrate that this metabonomic approach is a powerful tool with which to characterize changes in the metabolic level, and to facilitate the search for metabolic markers under certain physiopathological conditions, which may be used for early prognosis of FHF.
目前,代谢组学已广泛应用在药物毒性试验、功能基因组、临床诊断、临床治疗、疗效评估、营养学和分子流行病学等领域,但仍处在初步研究阶段尤其是在分析方法方面,怎样借助相关的分析手段建立一种合理的分析方法仍是一个急待解决的难题。
本论文构建了基于GC/TOF-MS小鼠血浆代谢组学分析方法,以及合适的数据处理、模式识别和统计学分析方法,并应用于D-GalN/LPS诱导的暴发性肝衰竭小鼠模型血浆代谢物谱分析,寻找相关代谢标志物,为暴发性肝衰竭的早期诊断奠定基础。
一、基于GC/TOF-MS的小鼠血浆代谢组学分析方法的建立
本实验在相关文献基础上,进一步考察不同有机溶剂的配比对内源性代谢产物提取效率的影响,并分别将水相、氯仿相以及水和氯仿混合相吹干衍生化进行GC/TOF-MS分析,利用相关统计学方法,研究了不同有机相分层后,各层对代谢物提取的影响,实验结果表明：同时加入氯仿、水和甲醇分层后,将水和甲醇混合相吹干衍生化,并通过GC/TOF-MS分析,得到总离子流色谱图中,无论是峰的个数还是峰的强度都优于其它方法。
将总离子流色谱图中每个色谱峰,借助AMDIS软件去卷积,通过NIST05标准谱库进行检索。结果显示,共监测到总代谢物峰269个,通过与标准谱库比对,55个谱峰相似度在700以上,认为是比较可靠的代谢物。并在检测到的55个代谢产物中任意选取18种,以核糖醇作为内标,考察基于GC/TOF-MS技术的稳定性,实验结果表明,系统稳定性、制样稳定性以及4℃存放一周的稳定性,其变异系数均在15以内,证明此方法稳定可行。在此方法基础上,利用氨基酸和单糖的标准品对相关峰进一步指认,建立了基于GC/TOF-MS方法的大鼠血浆内源性代谢物谱库,总共包含38个代谢产物,为以后血浆中GC/TOF-MS代谢物谱图的鉴定提供方便,提高工作效率。
二、利用已建方法进行D-氨基半乳糖/内毒素诱导的暴发性肝衰竭小鼠模型代谢组学分析
本实验将12只雄性BALB/c小鼠,随机分为D-Gal/LPS诱导的FHF组(6只)和对照组(6只)。根据质谱数据,监测到最多267个峰,其中52个被证实为糖、氨基酸、脂肪酸和有机酸等内源性代谢物。52个代谢物中,49个被识别而其他3个尽管未被识别但在两组之间有明显差异。这些物质广泛参与了机体的物质代谢和能量代谢的生化过程。
根据GC/TOF-MS色谱,D-Gal/LPS处理后血浆氨基酸水平发生了明显变化。与对照组比较,所识别的16种氨基酸在FHF组含量均有升高,除色氨酸的升高无统计学意义外,其余15种氨基酸(丙氨酸、缬氨酸、异亮氨酸、甘氨酸、丝氨酸、苏氨酸、脯氨酸、亮氨酸、天冬氨酸、苯丙氨酸、谷氨酰胺、谷氨酸、鸟氨酸、赖氨酸和酪氨酸)均明显升高,血浆中总氨基酸和生糖氨基酸的含量也明显升高。另外,FHF组BCAA/AAA值明显降低。
与对照组比较,非氨基有机酸中FHF组p-羟丁酸(HB)、延胡索酸、苹果酸、2,3,4-三羟基丁酸、尿酸等明显升高,而烯醇式丙酮酸(EP)、琥珀酸、十四烷酸明显降低。碳水化合物中除半乳糖和D-阿卓糖明显降低外,包括葡萄糖在内的其他化合物均无明显变化。另外,FHF组血浆磷酸、γ-氨基丁酸(GABA)、5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA)水平升高,而尿酸、软脂酸、硬脂酸含量均无异常变化。
依据血浆代谢物水平变化及D-Gal/LPS损伤肝脏的机制,所涉及代谢途径包括糖异生、糖酵解、酮体产生、三羧酸循环和尿素循环等。结果显示,与对照组比较,FHF组糖异生和糖酵解途径无明显变化,但酮体产生、三羧酸循环和尿素循环受到抑制。尽管几乎所有氨基酸水平升高,但只有少数如天冬氨酸、苯丙氨酸和酪氨酸促进了糖异生。
由于生化途径互相联系、高度调节、错综复杂,而且平均化处理的过程中会丢失信息,代谢组学的数据处理较为困难。诸如主成分分析等多变量统计学分析方法可以通过将大量的数据信息集中于一个特定数据组来减少数据复杂性。通过对两组小鼠血浆样本的GC/TOF-MS数据进行主成分分析,可以看出FHF组和对照组存在聚类特征且两组间存在明显的类别差异,使用SIMCA-P软件发现,代谢产物中5-HIAA、葡萄糖、HB和磷酸是对两个实验组的差异贡献值最大。
总之,本论文构建了基于GC/TOF-MS的小鼠血浆样本代谢组学分析方法,通过方法学考察表明该方法具有较好的实验精密度、线性和稳定性。并将该分析方法应用于D-氨基半乳糖／内毒素诱导的暴发性肝衰竭小鼠模型。通过与对照组比较,在D-Gal/LPS诱导的FHF小鼠模型,许多血浆代谢物水平发生了明显变化。而且,FHF早期的代谢变化中酮体生成、TCA和尿素循环受到抑制,但糖异生和糖酵解途径无明显变化。PCA数据分析显示5-HIAA、葡萄糖、HB和磷酸水平的组合是暴发性肝衰竭的潜在可靠标志物。以上结果说明这种方法可作为有力工具用以了解病理生理条件下代谢物水平的变化和进行相关代谢组学研究、寻找代谢标志物,并可用于暴发性肝衰竭的早期诊断。
Metabonomics has been increasingly highlighted in "omics" technology, which is to study the systematic changes of all the metabolites in response to external stimuli. Metabonomics is the extension of genomics and proteomics, reflecting the biological event that has occurred. Metabonomics research, in addition to the changes of drug metabolism themselves, centers on the drug-induced changes of endogenous metabolites, which can shed light on the process of diseases comprehensively and offer new ideas about disease prevention and diagnosis.
Currently, metabonomics has been widely used in toxicology, genomics, clinical diagnosis, clinical treatment, assessment of treatment, nutrition and molecular epidemiology, most of which are still in the preliminary research stage, especially in the analytical method. How to establish a rational method is still an urgent problem to be solved.
A metabonomic method, based on GC/TOF-MS of endogenous metabolites in small rat serum, as well as on the appropriate data processing, pattern recognition and statistical analysis methods, has been successfully applied to D-GalN/LPS-induced mouse model of fulminant hepatic failure(FHF) and facilitate the search for metabolic markers that, if linked to substance pathways, may also be used for early prognosis of FHF.
Firstly, a metabonomic method was established based on GC/TOF-MS of endogenous metabolites in plasma of mice.
plasma metabolite extraction conditions based on relevant literatures were developed, The extraction efficiency of different organic solvents, singly and in combination, was investigated to optimize the extraction condition of endogenous metabolites.
The result of the method,500μL of methanol,20μL of ribitol,450μL of pure water and 250μL of chloroform were added in Plasma samples(100μL) simultaneously, showed that its number and intensity of peak surpassed those of other methods By relevant statistical analysis.
On the basis of the mass spectral database(NIST05) and AMDIS software,269 peaks were detected in plasma samples, the similarity of 48 of which was above 7000 and confirmed to be endogenous metabolites in the TIC detected by GC/TOF-MS.
Eighteen endogenous metabolites were selected to investigate the system stability based on GC/TOF-MS technology at random from forty-eight metabolites.The result indicated most of the metabolites demonstrated good reproducibility, with intra-day, inter-day and stored in 4℃precision values below 15%. The relevant peaks were confirmed by standard compounds of monosaccharides and amino acids. The metabolites libraries of plasma were thus established, including 38 metabolites in the plasma library. These database can facilitate increasing the speed and accuracy of the peaks'identification in TIC.
Secondly, Metabonomic Analysis for D-GalN/LPS-induced mouse model of Fulminant Hepatic Failure(FHF).
12 male BALB/c mice were housed in a standard animal laboratory with a 12 h light-dark cycle and were provided with water and standard mouse chow ad libitum. The animals were randomly divided into GalN/LPS-induced FHF(n=6) and control(n=6)groups.
On the basis of the mass spectral database,267 peaks were detected,52 of which were confirmed to be endogenous metabolites, such as sugars, amino acids, fatty acids, and organic acids, among other compounds. Of the 52 metabolites,48 were identified, whereas the others were unidentified metabolites in which significant differences were noted between the FHF group and the controls. These substances have been implicated in multiple biochemical processes, including energy and substance metabolism.
Marked changes in plasma amino acids were observed on GC/TOF-MS chromatograms following GalN/LPS treatment. Though 16 amino acids were identified in total, amino acid concentrations were generally elevated in the FHF group, with significant differences in the levels of 15 amino acids(alanine,valine, isoleucine, glycine, serine, threonine, proline, leucine,aspartate, phenylalanine, glutamine, glutamate, ornithine,lysine, and tyrosine), with the exception of tryptophan, in which the increase was not statistically significant. The concentrations of total amino acids and gluconeogenic amino acids also increased. Furthermore, the ratio of BCAA to AAA (valine+leucine+isoleucine)/(tyrosine+phenylalanine+tryptophan) was significantly reduced in the FHF group.
Compared with the control group,β-hydroxybutyrate(HB), fumarate, aminomalonic acid, malate and uric acid were elevated in the treatment group. Interestingly, levels of enolpyruvate(EP), succinate, and tetradecanoic acid decreased significantly among the nonamino-organic acids identified in FHF mice. Although there was a noted decrease in galactose and D-altrose in these mice, no significant differences in other carbohydrates, including glucose, were noted. Elevated phosphate,γ-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT), and 5-hydroxyindol-eaceti acid(5-HIAA)levels were also observed after GalN/LPS treatment. Levels of urea, palmic acid and stearic acid were similar in both groups.
based on changes in the levels of intermediates in the FHF mice and the mechanisms underlying GalN/LPS damage to the liver, the pathways included gluconeogenesis, glycolysis, production of ketone bodies, the tricarboxylic acid(TCA) and urea cycles, among others. Whereas no significant differences between the fasted control and treated mice were noted in the gluconeogenic and glycolytic pathways, the production of ketone bodies, the TCA cycle, and the urea cycle were inhibited in the FHF mice. Although the levels of almost all amino acids were elevated in these mice, only a minority(e.g., aspartate, phenylalanine, and tyrosine) contributed to gluconeogenesis.
Interpretation of the metabolite level data is difficult, not only because biochemical pathways are linked and highly regulated but also because information is lost in the process of averaging. Analysis methods, such as PCA, reduce data complexity by focusing on the information content of a given data set, while maintaining most of the information in a few dimensions. Analysis with SIMCA-P and PCA softwares indicated distinct clustering was observed between the FHF group and the control group.5-HIAA, glucose, HB, and phosphate are the parameters with the highest weights, and thus they had the greatest impact on each of the principal components in each experimental group.
In all, based on GC/TOF/MS metabonomic method was establishped and applied for GalN/LPS-induced FHF. By the analysis of the metabolic profiling of plasma, significant differences in the plasma levels of many metabolites were noted, compared with those of the controls in the model of GalN/LPS-induced FHF. Furthermore, the earliest metabolic perturbations detected included the inhibition of ketogenesis and the TCA and urea cycles, with no significant changes in the gluconeogenic and glycolic pathways. PCA data analysis suggests that a combination of 5-HIAA, glucose, HB, and phosphate concentrations in the plasma is a potential marker for FHF. The above results demonstrate that this metabonomic approach is a powerful tool with which to characterize changes in the metabolic level, and to facilitate the search for metabolic markers under certain physiopathological conditions, which may be used for early prognosis of FHF.
引文
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