代谢物组学在医药研究中的成功实践

代谢物组学作为后基因时代的一种全新的组学技术。其主要以现代系统生物学为理论基础,以生物体液为研究对象,以现代谱学分析理论和生物样品制备方法为技术支撑,集中生物体内低分子量化学组分进行全息分析和海量数据挖掘,最终明晰机体生物学变化的本质。代谢物组学在功能基因组学、病理生理学、药理毒理学等方面都有着广泛的应用前景。本文以代谢物组学概念化的提出为切入点,着眼于代谢物组学的宽口径应用领域,重点概述代谢物组学在医药领域的成功实践,并对代谢物组学的未来发展做初步构想代谢物组学在功能基因组学、病理生理学、药理毒理学等方面都有着广泛的应用前景。     

植物内生微生物及其“暗物质”开发技术的研究进展

植物内生微生物包含内生菌、内生细菌、内生真菌、内生放线菌、内生古细菌等领域,其"暗物质"是指尚未被开发的菌种资源、遗传资源和代谢物资源。根据对宿主植物的作用,可以将内生微生物分为共生内生微生物、有益内生微生物和潜在的病原微生物三类。内生微生物在植物促生、提高植物抗逆性、提高植物的环境适应性、影响植物代谢物合成、参与被污染环境的修复等方面具有广泛的应用。具有生理活性的次级代谢产物的开发和利用是植物内生微生物研究的另一个热门领域,但由于大多数不能被分离培养、次级代谢物的分离分析技术不足等原因,使得大量的植物内生微生物资源仍处于未知状态。表面消毒技术、分离培养技术的改进,"多-组学"技术的联合使用和次级代谢物分离分析技术的发展极大促进了植物内生微生物的研究。在此基础上,开展微生物组研究和多单位多地域的协同研究将会进一步加快植物内生微生物资源的研究、发掘和利用。以上述内容为基础,本文综述了植物内生微生物的功能及其代谢产物研究进展,当前植物内生微生物研究的分离培养方法和组学技术,并在文章最后对该领域的研究方向提出了建议。     

功能代谢组学技术在微生物研究中的应用

功能代谢组学是以代谢组学技术发现关键代谢物为基础,结合体内体外实验和分子生物学等技术手段,研究差异代谢物及相关蛋白、酶和基因的功能,从而揭示生物体内在的分子调控机制。功能代谢组学技术具有精准识别关键调控代谢物及其相关基因或酶的特性,近年来在微生物相关疾病的防控和工业化生产等方面受到了广泛的关注。本文介绍了功能代谢组学技术的分析流程、相关研究方法与平台及其在微生物研究方面的应用,其中重点阐述了真核、原核以及病毒微生物的代谢特性、调控靶点及相关防控策略等。最后,提出功能代谢组学研究在未来面临的问题与挑战,为后续功能代谢组学的研究与发展提供新的思路。     

功能代谢组学技术在微生物研究中的应用

功能代谢组学是以代谢组学技术发现关键代谢物为基础,结合体内体外实验和分子生物学等技术手段,研究差异代谢物及相关蛋白、酶和基因的功能,从而揭示生物体内在的分子调控机制。功能代谢组学技术具有精准识别关键调控代谢物及其相关基因或酶的特性,近年来在微生物相关疾病的防控和工业化生产等方面受到了广泛的关注。本文介绍了功能代谢组学技术的分析流程、相关研究方法与平台及其在微生物研究方面的应用,其中重点阐述了真核、原核以及病毒微生物的代谢特性、调控靶点及相关防控策略等。最后,提出功能代谢组学研究在未来面临的问题与挑战,为后续功能代谢组学的研究与发展提供新的思路。     

代谢组学技术及其在临床研究中的应用

在后基因组时代, 系统生物学研究成为人们关注的焦点。转录组学、蛋白质组学等功能基因组学研究方法可同时检测药物或其他因素影响下大量基因或蛋白质的表达变化情况, 但这些变化不能与生物学功能的变化建立直接联系。代谢组学方法则可为代谢物含量变化与生物表型变化建立直接相关性。代谢组学研究的目的是定量分析一个生物系统内所有代谢物的含量, 进行全面代谢物分析需要分析化学技术的支撑, 核磁共振和基于质谱的分析技术是代谢组学研究的两种主要技术手段。代谢组学研究可产生大量数据信息, 对这些数据进行分析离不开化学统计学的应用, 比如主成分分析、多维缩放、各种聚类分析技术以及功能差异分析等。文章综述了近年来代谢组学分析技术及数据分析技术的研究进展, 在此基础上, 对代谢组学在临床研究及临床前研究中的应用研究进展进行了综述。对疾病代谢表型图谱的研究有助于人们了解疾病发生、发展以及致死的机制; 在临床条件下, 这些代谢图谱可以作为疾病诊断、预后以及治疗的评判标准。代谢物组成的变化是毒物胁迫对机体造成的最终影响, 利用代谢组技术可以直接反映毒物对机体的影响。质谱技术、核磁共振技术的应用使得药物筛选过程可以快速完成, 并有助于实现个性化用药。此外, 利用代谢组学技术还可以进行已知酶的新活性研究, 也可以研究未知酶。     

基于代谢组学技术的植物抗病相关代谢物研究进展

植物受到病原真菌侵染时往往通过调节体内代谢物的产生来增强自身抗性,代谢组学技术是研究植物抗病相关代谢物的重要工具。指认植物抗病相关代谢物不仅利于深入探讨其抗病机制,还可与其他组学技术结合,辅助抗性品种鉴定和抗病品种培育。该研究对近年来国内外有关基于代谢组学技术指认植物抗病相关代谢物的流程、已发现的抗病相关代谢物及其作用机制的研究进展进行综述,并探讨了目前应用代谢组学技术研究植物抗病相关代谢物过程中面临的挑战。     

代谢物组学及其在微生物研究中的应用

代谢物组学(metabolomics)是继基因组学(genomics)、蛋白质组学(proteomics)后发展起来的一门新学科。对代谢物组学的含义,研究方法及流程,特别是其在微生物中的应用进行了介绍,包括使用代谢物组学中的NMR技术研究微生物在降解环境污染物中的作用;使用代谢物组学技术研究微生物代谢通量,从而在分析代谢通量的基础上通过代谢工程改变代谢通量,提高目的产物的得率;确定所获得基因库中沉默基因的功能;运用代谢物组学分析方法阐明生物体系对于环境变化的响应,从而协助我们确定最佳的取样时间及最佳分析组织,设计实验。随后简要对代谢物组学发展动态进行了展望。     

代谢组学技术在微藻研究中的应用

代谢组学(metabolomics/metabonomics)是系统生物学的重要组成部分之一,主要通过分析生物体受环境刺激、病理生理或基因变异等因素引起的内源性小分子代谢物变化来研究其生理功能与代谢之间的关系,进而揭示代谢物变化背后的代谢调控机制与机理.代谢组学技术具有灵敏度高、选择范围广和分析速度快等特点,逐渐在微藻...     

基于质谱信号的代谢物鉴定:生物信息学的机遇与挑战

液相色谱-质谱联用是目前代谢组学研究中广泛采用的分析技术。随着海量质谱数据的产生,解析质谱信号所对应的代谢物分子显得越来越重要。由于代谢物中分子成分复杂多变,其质谱信号具有特定的数据分布特点。因此,开发适应这一特点的分析软件,扩展通用标准谱图库和代谢物数据库,增加代谢物的鉴定率是当前代谢组学研究的关键任务。本文主要介绍近年来基于质谱信号的代谢物鉴定研究领域的基本思路和最新进展。     

基于质谱技术的细胞代谢组学研究进展

细胞代谢组学作为一个新兴领域,能解决基本的生物学问题,还能观察细胞内的代谢情况。细胞代谢物浓度可以近似地反映一个组织、器官或细胞的表型。随着代谢组学的发展,以质谱分析为基础的代谢组学技术研究细胞的代谢物,其灵敏度高、分辨率好,能进行多组分的检测,并能获取分子的结构信息,这有利于细胞生物学的研究。该文结合目前代谢组学的技术,对细胞代谢物研究的意义及基于质谱技术的细胞代谢组学的应用进行了综述。     

Highly sensitive and specific gas chromatographic-tandem mass spectrometric method for the determination of trace amounts of antipyrine metabolites in biological material

A highly sensitive and specific gas chromatographic-tandem mass spectrometric method was developed for the determination of the antipyrine (INN: phenazoe) metabolites, norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine, in biological material. Deuterated analogues of the metabolites were used as internal standards. The method has a limit of quantitation of 5 ng per sample for the determination of norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine with coefficients of variation of 19.4, 14.6 and 20.7%, respectively. Precision and accuracy are good over the whole range measured (5–500 ng/sample) with a coefficient of variation, respectively error of determination ⩽20%. Due to its high sensitivity the method can be used to study the formation of these metabolites in microsomal preparations containing only 100 μg of protein.     

Studies of the metabolism of the antihistaminic drug dimethindene by high-performance liquid chromatography and capillary electrophoresis including enantioselective aspects

A reversed-phase high-performance liquid chromatography method for the determination of dimethindene and its main metabolites N-demethyldimethindene, 6-hydroxydimethindene and 6-hydroxy-N-demethyldimethindene in human urine was developed. The assay was also applied to the quantification of dimethindene-N-oxide in rat urine. Conjugates of the hydroxylated metabolites were determined after enzymatic deconjugation. Moreover the direct determination of dimethindene and its metabolites without prior extraction from urine was performed by capillary electrophoresis. The direct simultaneous determination of the enantiomers of dimethindene and N-demethyldimethindene was achieved on a Chiralcel OD column. Urinary data after oral administration of dimethindene are presented. The assays were used to study dimethindene and it metabolites in urine upon oral administration of the drug to rats and human volunteers.     

Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection

A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidiized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The metthod was applied to the determination of AJ and the metabolites in rat plasma samples.     

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible.

     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

Mass fragmentographic quantitation of ethotoin and some of its metabolites in human urine

A method for the determination of ethotoin and its p-hydroxylated and dealkylated metabolites in urine has been developed. Ethotoin and the metabolites were extracted from acidified urine with ethyl acetate and silylated before injection into a combined gas chromatograph—mass spectrometer. Four partly identified metabolites were recorded, but their exact quantitation was not possible as pure reference substances were not available.The limit of sensitivity was far below the amounts of ethotoin and of its metabolites found in urine from patients treated with therapeutic dozes of ethotoin.     

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate–methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [¹⁸F]fluoroethylflumazenil in routine clinical analysis.     

Determination of chlordiazepoxide,diazepam, and their major metabolites in blood or plasma by spectrophotodensitometry

An analytical procedure was developed for the determination of chlordiazepoxide, diazepam and their major metabolites in blood or plasma. Demoxepam, a metabolite of chlordiazepoxide, is determined by spectrofluorometry after selective extraction. The remaining compounds are determined by spectrophotodensitometry after thin-layer chromatographic separation.The sensitivity limit of the spectrofluorometric determinationn of demoxepam is 0.1 to 0.2 μg while that of the spectrophotodensitometric determination of chlordiazepoxide, diazepam and their N-desmethyl metabolites is 0.05 to 0.2 μg. The sensitivity and specificity of the assay renders it suitable for monitoring plasma levels of chlordiazepoxide and its major metabolites following single or chronic oral administration of chlordiazepoxide hydrochloride. The sensitivity limit for diazepam and nordiazepam, its major metabolite, renders the assay useful only for the determination of plasma concentrations resulting from high dosage of diazepam. The assay was used to determine chlordiazepoxide and its metabolites following oral administration of Librium. The data showed a significant correlation to those obtained on the same specimens by differential pulse polarography and by radio-immunoassay.     

Gas chromatographic determination of aromatic acid metabolites of phenylalanine in brain

A method for the determination of the aromatic acid metabolites of phenylalanine in brain by gas-liquid chromatography is described. Procedures were developed for the extraction and purification of the metabolites, the preparation of their trimethylsilyl derivatives, the separation and identification of these derivatives by gas-liquid chromatography, and the quantification of the metabolites by employing the internal reference standards phenylvaleric and o-hydroxyphenylacetic acids with the detector molar response factors. The metabolites in the hyperphenylalaninemic brain were identified as the trimethylsilyl ester of phenylacetic, ester-ethers of mandelic and phenyllactic, and the ester-enol ether of the oxime of phenylpyruvic acid.