代谢组学技术在营养学研究中的应用

代谢组学可以系统性地研究生物体受到扰动或干预后整体代谢水平的变化。文章概述了代谢组学技术的生物样品采集与制备、分析技术、数据处理与统计分析、差异代谢物结构鉴定与代谢通路分析等方法与步骤及其在营养学研究领域中的应用,展望了代谢组学技术在营养学研究领域的应用前景,以期为下一步的研究提供参考。     

毛细管电泳质谱联用技术及其在代谢组学研究中的应用

毛细管电泳自20世纪80年代中后期迅速发展以来,是近几年分析化学领域中发展最为迅速的分离手段之一。毛细管电泳与质谱的联用使得分析范围更广,灵敏度更高,因此被广泛应用于生物样品的分析中。介绍了毛细管电泳质谱联用常用的接口技术,质量分析器及其在生物样品中的分离模式,综述了近年来毛细管电泳质谱联用技术在代谢组学中的应用。     

基于气相色谱-质谱联用技术的代谢组学方法在转植酸酶基因玉米代谢物指纹图谱分析中的应用

目的:建立气相色谱-质谱联用技术(GC-MS)的代谢组学方法,初步研究转基因株系与对照株系之间代谢物指纹图谱的差异性,为转基因作物安全的评价提供参考。方法:优化提取条件,考察色谱条件,并采用主成分分析(PCA)数据处理方法对转基因株系及对照进行模式识别。结果:优化了提取条件及色谱条件,建立了GC-MS的代谢组学方法,获得了小分子的代谢产物的表达谱,发现转基因与其对照之间呈现出显著性差异。结论:优化的GC-MS的代谢组学方法可以从代谢水平检测转基因作物,找出差异性,为转基因作物的检测与评价提供技术支持。     

代谢组学研究技术及其应用

介绍代谢组学的研究技术,主要包括核磁共振技术,色谱-质谱联用技术,同时介绍常用的数据处理方法和数据库.对目前代谢组学在医药领域、生物研究领域、资源环境,以及农业和食品领域的应用情况进行综述.     

气相色谱—质谱联用技术在植物激素分析中的应用

本文结合自己多年来工作的体会介绍了GC—MS技术如何用于植物激素分析和在分析过程中应注意的关键技术问题。     

两种提取溶剂对莲雾果实GC-MS代谢组学分析的影响

以台湾"黑珍珠"莲雾果实为实验材料,选择植物代谢组学研究中常用的两种提取溶剂对莲雾果实样品进行非靶标的代谢轮廓分析,研究两种提取溶剂对莲雾果实代谢组学分析的影响。利用气相色谱-质谱联用(GC-MS)技术对莲雾果实代谢产物进行分离鉴定,利用甲醇-水(A)法和甲醇-氯仿-水(B)法两种方法共鉴定出129个代谢产物;利用主成分分析法等比较了两种提取方法的提取效果,结果表明两种方法存在显著差异。从色谱峰总数来看,A法可检测到323个色谱峰,B法仅有251个,统计分析发现124个差异显著的代谢物质,其中有90个代谢物质的相对含量用A法提取显著高于B法提取。研究结果表明,甲醇-水溶剂提取的方法更适合于莲雾果实代谢组学的分析研究。     

两种提取溶剂对莲雾果实GC-MS代谢组学分析的影响

以台湾"黑珍珠"莲雾果实为实验材料,选择植物代谢组学研究中常用的两种提取溶剂对莲雾果实样品进行非靶标的代谢轮廓分析,研究两种提取溶剂对莲雾果实代谢组学分析的影响。利用气相色谱-质谱联用(GC-MS)技术对莲雾果实代谢产物进行分离鉴定,利用甲醇-水(A)法和甲醇-氯仿-水(B)法两种方法共鉴定出129个代谢产物;利用主成分分析法等比较了两种提取方法的提取效果,结果表明两种方法存在显著差异。从色谱峰总数来看,A法可检测到323个色谱峰,B法仅有251个,统计分析发现124个差异显著的代谢物质,其中有90个代谢物质的相对含量用A法提取显著高于B法提取。研究结果表明,甲醇-水溶剂提取的方法更适合于莲雾果实代谢组学的分析研究。     

两种提取溶剂对莲雾果实GC-MS代谢组学分析的影响

以台湾"黑珍珠"莲雾果实为实验材料,选择植物代谢组学研究中常用的两种提取溶剂对莲雾果实样品进行非靶标的代谢轮廓分析,研究两种提取溶剂对莲雾果实代谢组学分析的影响。利用气相色谱-质谱联用(GC-MS)技术对莲雾果实代谢产物进行分离鉴定,利用甲醇-水(A)法和甲醇-氯仿-水(B)法两种方法共鉴定出129个代谢产物;利用主成分分析法等比较了两种提取方法的提取效果,结果表明两种方法存在显著差异。从色谱峰总数来看,A法可检测到323个色谱峰,B法仅有251个,统计分析发现124个差异显著的代谢物质,其中有90个代谢物质的相对含量用A法提取显著高于B法提取。研究结果表明,甲醇-水溶剂提取的方法更适合于莲雾果实代谢组学的分析研究。     

两种提取溶剂对莲雾果实GC-MS代谢组学分析的影响

以台湾"黑珍珠"莲雾果实为实验材料,选择植物代谢组学研究中常用的两种提取溶剂对莲雾果实样品进行非靶标的代谢轮廓分析,研究两种提取溶剂对莲雾果实代谢组学分析的影响。利用气相色谱-质谱联用(GC-MS)技术对莲雾果实代谢产物进行分离鉴定,利用甲醇-水(A)法和甲醇-氯仿-水(B)法两种方法共鉴定出129个代谢产物;利用主成分分析法等比较了两种提取方法的提取效果,结果表明两种方法存在显著差异。从色谱峰总数来看,A法可检测到323个色谱峰,B法仅有251个,统计分析发现124个差异显著的代谢物质,其中有90个代谢物质的相对含量用A法提取显著高于B法提取。研究结果表明,甲醇-水溶剂提取的方法更适合于莲雾果实代谢组学的分析研究。     

两种提取溶剂对莲雾果实GC-MS代谢组学分析的影响

以台湾"黑珍珠"莲雾果实为实验材料,选择植物代谢组学研究中常用的两种提取溶剂对莲雾果实样品进行非靶标的代谢轮廓分析,研究两种提取溶剂对莲雾果实代谢组学分析的影响。利用气相色谱-质谱联用(GC-MS)技术对莲雾果实代谢产物进行分离鉴定,利用甲醇-水(A)法和甲醇-氯仿-水(B)法两种方法共鉴定出129个代谢产物;利用主成分分析法等比较了两种提取方法的提取效果,结果表明两种方法存在显著差异。从色谱峰总数来看,A法可检测到323个色谱峰,B法仅有251个,统计分析发现124个差异显著的代谢物质,其中有90个代谢物质的相对含量用A法提取显著高于B法提取。研究结果表明,甲醇-水溶剂提取的方法更适合于莲雾果实代谢组学的分析研究。     

Gas chromatography-mass spectrometry of oleanane- and ursane-type triterpenes—application to Chenopodium quinoa triterpenes

Nine trimethylsilylated pentacyclic triterpenes were separated by GLC on an OV-101 column employing temperature programming. Characteristic retro-Diels-Alder fragmentation was observed in their mass spectra. The fragmentation patterns allowed individual characterization except for certain isomers which, nevertheless, were resolved by GLC, thus permitting their identification. Oleanolic acid and hederagenin were confirmed to be major triterpenes of Chenopodium quinoa seed saponins.     

Determination of putrescine in brain tissue using gas chromatography-mass spectrometry

We used gas chromatography-mass spectrometry to assay putrescine in minute regions of single rat brains. Acid extraction, partial purification on Amberlite CG 120, and derivatization with pentafluoropropionic anhydride preceded the gas chromatography-mass spectrometry. A moving-needle solventless system and a direct inlet system were also used to increase sensitivity. Putrescine was measured accurately at the picomole level; the mean concentration of this polyamine in five regions of rat brain found by this method was 2.7-3.8 times higher than reported by other researchers.     

Gas chromatography-mass spectrometry (GC-MS) techniques for metabolic flux analysis of the Bifido shunt pathway

The presence of the Bifido shunt in Bifidobacterium is predicted to lead to the uptake and metabolic conversion of fructose to acetate and lactate. We propose an approach to quantifying the carbon flux through the Bifido shunt by measuring specific ¹³C-labeled carbohydrate-derived isotopomers by gas chromatography-mass spectrometry (GC-MS). The techniques described may provide an alternative approach for determining the in vitro prebiotic potential of dietary oligosaccharides.     

Gas chromatography-mass spectrometry (GC-MS) techniques for metabolic flux analysis of the Bifido shunt pathway

The presence of the Bifido shunt in Bifidobacterium is predicted to lead to the uptake and metabolic conversion of fructose to acetate and lactate. We propose an approach to quantifying the carbon flux through the Bifido shunt by measuring specific ¹³C-labeled carbohydrate-derived isotopomers by gas chromatography-mass spectrometry (GC-MS). The techniques described may provide an alternative approach for determining the in vitro prebiotic potential of dietary oligosaccharides.     

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes

A new technique for the conversion of 2-acetylaminofuorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofuorene and N-hydroxy-2-acetylaminofuorene.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - DMF dimethylformamide - El electron impact ionization - FBS fetal bovine serum - GC-MS gas chromatography-mass spectrometry - MtBSTFA N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide - MU methylene unit - N-OH-2-AAF N-hydroxy-2-acetylaminofluorene - 4,4-OH-BP 4,4-hydroxybiphenyl - tBDMS tert.-butyldimethylsilyl     

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.     

Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[²H₃]acetylneuraminic acid as an internal standard. M-COOTMS fragments at $\text{m}\text{z}$ 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at $\text{m}\text{z}$ 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10³, and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

Indole-3-acetic acid determination in cultured crown-gall and genetic tumour tissues by gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) was identified and quantified in three cultured tumour lines, which included crown-gall tissue of Datura (25.4 ng/g fresh wt.) and two genetic tumour lines of tobacco (4.6 and 8.0 ng/g fresh wt.). The analysis was carried out using a stable isotope dilution assay in combination with gas chromatography-mass spectrometry. This is the first unambiguous determination of endogenous IAA in genetic tumour tissues.     

玉竹的不同溶剂提取物中化学成分的GC-MS对比分析

建立了采用气相色谱-质谱技术分析压竹的不同溶剂提取物中化学成分的方法.分别用石油醚、正己烷和乙酸乙酯浸泡萃取玉竹,然后将萃取液于25℃、0.015 MPa下减压蒸馏浓缩,并对石油醚和正己烷的浓缩提取物进行了GC-MS对比分析.实验结果表明,石油醚提取物中含有11种芳香族化合物和13种其它组分,它们占总出峰面积的81.33%,其中,甲苯和1,2-二甲苯为主要成分,含量分别达到了9.38%和13.38%.正己烷提取物中含有37.03%的柠檬油精(D-limonene),而石油醚提取物中未发现此柠檬油精(D-limonene),在正己烷提取物中确认了19种化合物,它们占总出峰面积的95.99%.乙酸乙酯不适合直接做浸泡萃取玉竹精油的提取溶剂.