基于GC/MS技术的脂肪酸代谢轮廓谱方法和应用研究
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摘要
包含脂肪酸代谢在内的脂质代谢循环,一直以来就是糖尿病、心血管病等重大疾病发生机理的研究热点。本文采用GC/MS技术对脂肪酸代谢组学进行研究。建立了一种脂肪酸代谢轮廓谱方法,详细考察了血浆用量、衍生化方法、脂肪酸提取分离、内标/外标法选定等分析条件对定量结果的影响。该方法实现了对25种酯化脂肪酸(EFAs)和25种非酯化脂肪酸(NEFAs)同时定性,及其中15种EFAs和15种NEFAs的精确定量。方法学考察结果表明,该方法线性良好,相关系数大于0.997；日内,日间RSDs小于15%；脂肪酸加标回收率(高、中、低浓度)为80-110%。该法仅需用10μL血浆或血清样本,较文献报道用量(150-300μL)大为减少。
将建立的脂肪酸代谢轮廓谱方法用于糖尿病、糖尿病肾病的临床代谢组学研究。定量测定了血浆中15种EFAs和15种NEFAs.基于所得定量数据,对脂肪酸总体水平、各类脂肪酸水平、各个脂肪酸水平以及多个脂肪酸含量比值等变化进行分析,探讨了脂肪酸代谢水平变化与糖尿病、糖尿病肾病发病机理之间的关系。采用PLS-DA等模式识别技术,对所得脂肪酸测定结果进行聚类分析,各组样本聚集成类,体现了疾病发展趋势,以上结果验证了脂毒性假说。基于定量结果指认了中医学分型的潜在疾病生物标记物,根据不同中医分型样本中潜在生物标志物水平变化趋势,初步认为,多不饱和脂肪酸是体现贯穿“消渴证”始终的“血瘀”、“湿浊”、“痰阻”、“水停”等“证”的部分物质基础。
营养干预是目前预防神经管畸形(NTDs)发病的主要手段,但其预防发病的机理目前尚不清楚。本文对营养干预前后的育龄期妇女进行了血清脂肪酸代谢轮廓谱对比分析。基于血清中EFAs和NEFAs定量结果,初步探讨了营养干预可能对育龄期妇女脂肪酸代谢造成的影响,以及这些影响与NTDs发病相关的代谢调节途径。
最后,还将GC/MS技术用于微生物发酵液和中药挥发油为代表的其他生物复杂样品分析,实现了微生物发酵液中木糖醇、木糖、葡萄糖的同时定性、定量,该法可用于微生物发酵法生产木糖醇的过程监控。
Lipid metabonomics including fatty acids metabolism is always the hottest topic in the pathology researching field of metabolic syndrome such as diabetes, cardiovascular disease, etc. A new metabonomic profiling method was developed for simultaneously qualitative and quantitative analysis on esterified and nonesterified fatty acids (EFAs and NEFAs) in blood (plasma or serum). Merely 10μL of plasma/serum was required. The pretreatment of the sample was simple without disposing the protein.15 FAs(both EFAs and NEFAs) in plasma were precisely quantified. Gas Chromatography-Mass Spectrometry (GC-MS) was used in the study. The quantities of the analytes varied in abundance over 3 orders of magnitude. The established method showed good accuracy. The curve correlation coefficients were all above 0.997. The RSDs of precision for all compounds were below 15% and recoveries were all between 80～110%. PLS-DA modal was used in the data management. The method was later applied into clinical metabolomics study ("Health-disease") and nutritional metabonomics ("nutrition intervention-control") study.
The metabonomic profiling method was then applied to plasma FAs quantification in diabetes mellitus (DM) and diabetic nephropathy (DN) study. The quantification results were given by Modern Medicine classification and Traditional Chinese Medicine classification. Based on the data anaylsis, the FAs'biological significances related with the pathology of DM and DN were discussed in detail. The results support the hypothesis of lipitoxicity, which is an important thesis in the pathology of DM. Potential biomarkers (both NEFAs and EFAs) for different views of classification were identified. Their biological may had important effection during every adjacent stage of developing process of DM and DN; PLS-DA analysis using the SIMCA-P software package allowed the different course of disease to be distinguished.
"Xiaoke Zheng" is the counterpoint conception of DM and DN in Traditional Chinese Medicine (TCM). And "Zheng" is a very important conception in the Traditional Chinese Medicine (TCM). Its meaning is so complicated and comprehensive that cannot be represented by a single metabolite indicator. In this research, some pathological phenomena of "Xiaoke Zheng", such as "xueyu", "shizhuo", "tanzu", "shuiting", etc., have been discussed on the basis of EFAs and NEFAs quantification results.
Nutrition intervention (NI) is the common means for Neural tube defects (NTDs) prevention. We study the serum EFAs and NEFAs metabolic profiling of Child-bearing age women by using GC/MS method, The subjects were random divided into two groups:NI group and control group. The mean levels of EFAs and NEFAs of these two groups were reported. PLS-DA allowed the different group to be distinguished, which means NI has great effects on the metabolism of fatty acids. Based on the quantification results,6 FAs (both EFAs and NEFAs) were recognized as potential biomarkers. And the relationship between NI and the FAs'concentration variation had been discussed to illustrate the potential impacts on the incidence of NTDs.
Other complicated matrixes were also been explored based on GC-MS methods, such as: bio-fermentation liquids and volatile oil of two classic TCM. A new analytical method was put forward for the quantification of polyols in bio-fermentation liquids. The method could be used in the monitoring of polyol production process. The efficacy similarities and differences of Traditional Chinese Medicine Danggui and Chuanxiong were studied through comparation of their volatile oil composition. NIST spectrum library retrieving was used to quality the compounds in the two TCMs'volatile oil. Their relative quantificaions were conducted through integration of the peak area. The two TCMs'functions were discussed based on the analysis results.
将建立的脂肪酸代谢轮廓谱方法用于糖尿病、糖尿病肾病的临床代谢组学研究。定量测定了血浆中15种EFAs和15种NEFAs.基于所得定量数据,对脂肪酸总体水平、各类脂肪酸水平、各个脂肪酸水平以及多个脂肪酸含量比值等变化进行分析,探讨了脂肪酸代谢水平变化与糖尿病、糖尿病肾病发病机理之间的关系。采用PLS-DA等模式识别技术,对所得脂肪酸测定结果进行聚类分析,各组样本聚集成类,体现了疾病发展趋势,以上结果验证了脂毒性假说。基于定量结果指认了中医学分型的潜在疾病生物标记物,根据不同中医分型样本中潜在生物标志物水平变化趋势,初步认为,多不饱和脂肪酸是体现贯穿“消渴证”始终的“血瘀”、“湿浊”、“痰阻”、“水停”等“证”的部分物质基础。
营养干预是目前预防神经管畸形(NTDs)发病的主要手段,但其预防发病的机理目前尚不清楚。本文对营养干预前后的育龄期妇女进行了血清脂肪酸代谢轮廓谱对比分析。基于血清中EFAs和NEFAs定量结果,初步探讨了营养干预可能对育龄期妇女脂肪酸代谢造成的影响,以及这些影响与NTDs发病相关的代谢调节途径。
最后,还将GC/MS技术用于微生物发酵液和中药挥发油为代表的其他生物复杂样品分析,实现了微生物发酵液中木糖醇、木糖、葡萄糖的同时定性、定量,该法可用于微生物发酵法生产木糖醇的过程监控。
Lipid metabonomics including fatty acids metabolism is always the hottest topic in the pathology researching field of metabolic syndrome such as diabetes, cardiovascular disease, etc. A new metabonomic profiling method was developed for simultaneously qualitative and quantitative analysis on esterified and nonesterified fatty acids (EFAs and NEFAs) in blood (plasma or serum). Merely 10μL of plasma/serum was required. The pretreatment of the sample was simple without disposing the protein.15 FAs(both EFAs and NEFAs) in plasma were precisely quantified. Gas Chromatography-Mass Spectrometry (GC-MS) was used in the study. The quantities of the analytes varied in abundance over 3 orders of magnitude. The established method showed good accuracy. The curve correlation coefficients were all above 0.997. The RSDs of precision for all compounds were below 15% and recoveries were all between 80～110%. PLS-DA modal was used in the data management. The method was later applied into clinical metabolomics study ("Health-disease") and nutritional metabonomics ("nutrition intervention-control") study.
The metabonomic profiling method was then applied to plasma FAs quantification in diabetes mellitus (DM) and diabetic nephropathy (DN) study. The quantification results were given by Modern Medicine classification and Traditional Chinese Medicine classification. Based on the data anaylsis, the FAs'biological significances related with the pathology of DM and DN were discussed in detail. The results support the hypothesis of lipitoxicity, which is an important thesis in the pathology of DM. Potential biomarkers (both NEFAs and EFAs) for different views of classification were identified. Their biological may had important effection during every adjacent stage of developing process of DM and DN; PLS-DA analysis using the SIMCA-P software package allowed the different course of disease to be distinguished.
"Xiaoke Zheng" is the counterpoint conception of DM and DN in Traditional Chinese Medicine (TCM). And "Zheng" is a very important conception in the Traditional Chinese Medicine (TCM). Its meaning is so complicated and comprehensive that cannot be represented by a single metabolite indicator. In this research, some pathological phenomena of "Xiaoke Zheng", such as "xueyu", "shizhuo", "tanzu", "shuiting", etc., have been discussed on the basis of EFAs and NEFAs quantification results.
Nutrition intervention (NI) is the common means for Neural tube defects (NTDs) prevention. We study the serum EFAs and NEFAs metabolic profiling of Child-bearing age women by using GC/MS method, The subjects were random divided into two groups:NI group and control group. The mean levels of EFAs and NEFAs of these two groups were reported. PLS-DA allowed the different group to be distinguished, which means NI has great effects on the metabolism of fatty acids. Based on the quantification results,6 FAs (both EFAs and NEFAs) were recognized as potential biomarkers. And the relationship between NI and the FAs'concentration variation had been discussed to illustrate the potential impacts on the incidence of NTDs.
Other complicated matrixes were also been explored based on GC-MS methods, such as: bio-fermentation liquids and volatile oil of two classic TCM. A new analytical method was put forward for the quantification of polyols in bio-fermentation liquids. The method could be used in the monitoring of polyol production process. The efficacy similarities and differences of Traditional Chinese Medicine Danggui and Chuanxiong were studied through comparation of their volatile oil composition. NIST spectrum library retrieving was used to quality the compounds in the two TCMs'volatile oil. Their relative quantificaions were conducted through integration of the peak area. The two TCMs'functions were discussed based on the analysis results.
引文
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