Metabolites extraction

As in the other -omics, analyses may be directed at a specific metabolite, at all metabolites in a given system in a shot-gun approach, or at accessible groups of molecules in profiling experiments. In that also the technology varies. In addition, the chemistry of different metabolites is very heterogeneous since it involves hydrophobic lipids, hydrophilic carbohydrates, ionic inorganic species, and other secondary natural products and already the choice of solvent in metabolite extraction dictates which types of molecules will be present (Fig. 10.8). Therefore, total metabolome profiling is not possible, because no analytical method will be able to accommodate all the different molecule classes at once. 

Figure 3. TLC system used to separate trifiuralin and metabolites extracted from...

We employed various substrates to check for MFO in two bivalve species, a salt water mussel (Mytilus edulis) and a fresh water clam (Anodonta sp). Cytochrome P-450 was also studied. Organisms were exposed to 100 PPM Venezuelan crude in a stagnant system for up to one month. Enzyme assays were carried out with digestive gland 9000 g homogenates (17) and cytochrome P-450 analysis, with microsomes (21). The hydrocarbon substrates investigated included 1I+C-labelled benzo(a)pyrene, fluorene, anthracene, and naphthalene. The method used for separation of BP metabolites by thin layer radiochromatography has been described (7). The metabolite detection method for the other aromatic hydrocarbons was essentially the same except methylene chloride was used as metabolite extractant as well as TLC developer. Besides the hydrocarbon substrates, we also checked for other MFO reactions, N-dealkylase with C-imipramine (22) and 0-dealkylase with ethoxycoumarin (15). 

The stationary phase may be a solid or liquid on a solid support. The mechanisms responsible for distribution between phases include surface absorption, ion exchange, relative solubilities and steric affects . High performance liquid chromatography is a useful method for quinolizidine alkaloid analysis, especially when pure standards are available". This method was recently used for alkaloid metabolite extraction and analysis . A simple reversed-phase liquid chromatographic method has been developed for the simultaneous quantitation of four anticancerous alkaloids vincristine, vinblastine, and their precursors catharanthine and vindoline using a specific HPLC column . 

A dietary supplement is any product taken by mouth that contains a so-called dietary ingredient and whose label clearly states that it is a dietary supplement. The dietary ingredients in dietary supplements may include vitamins, minerals, herbs, and amino acids as well as substances such as enzymes, organ tissues, metabolites, extracts, or concentrates. Dietary supplements can be... 

Figure 24-6 Elution profile of 13N-containing metabolites extracted from liver 15 s after injection of 13NH3 into the portal vein of an anesthetized adult male rat. CIT, citrulline ARG SUC, argininosuccinate. From Cooper et al.89...

A study of the metabolism of SPI in pig liver was conducted. The polar character of the cysteyl derivatives makes them difficult to extract in chlorinated solvents (dichloromethane, chloroform). As a consequence, extraction with pure MeOH was considered, because it extracted both SPI and its cysteyl metabolites. However, it also extracted other biomolecules interfering with the metabolites. Extractions with pure MeCN were unsuccessful, since liver tissues tended to agglomerate in this medium. Only 40% of the cysteyl conjugates were then extracted. The property of water to disperse the liver tissue was used to develop extraction conditions with MeCN-water (90 10). A good dispersion of the liver was then obtained, and pollution by polar interfering compounds coextracted from the liver was limited. Acetonitrile was evaporated and MeOH was added... 

A typical metabolite profiling analysis involves the following steps (i) metabolite extraction that often has to be adapted on a case-by-case basis depending on the type of sample and analytical platform chosen (ii) sample preparation which may include partial purification and derivatization steps (iii) instrumental analysis of sample (iv) detection and quantification of metabolite signals to generate a data matrix that summarizes the detected signals and their intensity data and (v) statistical analysis of metabolite profiles (67). 

Owing to its relevancy in metabolite profiling analysis, several groups have investigated the suitability of different metabolite extraction procedures for GMO analysis. Selective extraction techniques, such as supercritical fluids... 

The purpose of this review is to present an overview of these secondary metabolites extracted from Astragalus spp., the newer methodology used for their isolation and structure elucidation and the biological activities of these compounds reported up to the beginning of 2000. 

DANS-based comparisons of daf-22 and wild-type metabolite extracts revealed several partial structures representing compounds produced only by wild type but not daf-22 worms, including several previously unknown compounds. These compounds represented far <0.1% of the entire metabolite mixture and therefore further characterization through HSQC or HMBC was not possible based on spectra of the unfractionated metabolite extracts. However, the differentially produced compounds were easily identified after partial... 

Figure 6 GC-TOF-MS analysis of a complete methanol-chloroform-water plant extract. The injection of a plant metabolite extract without separation of polar and hydrophobic phase is shown. Most of the metabolite compound classes are found in such a chromatogram. Reproduced with permission from M. Glinski W. Weckwerth, Mass Spectrom. Rev. 2006,25 (2), 173-214. Copyright Wiley-VCFI Verlag GmbFI Co. KGaA.

After metabolite extraction, enrichment of target metabolites or other polishing steps such as desalting or solvent exchange may be necessary. One possibility for such a cleanup is solid-phase extraction (SPE). The principle... 

Different analytical chemistry methods are used for analysis of the metabolome (Figure 3). Direct-infusion mass spectrometry (DI-MS) on both, low and (ultra)high resolution MS, infuses raw metabolite extracts in the mass spectrometer without prior chromatography or electrophoretic separation and often uses high-resolution mass spectrometers. This method offers fast analysis with low duty cycles however, isomeric and isobaric substances cannot be... 

Hydrolysis of the conjugated PAH metabolite extraction of the free metabolite into n-hexane concentration and methylation with methyl iodide extraction of methylated product... 

Trichoharzin (83) has been isolated from a culture of a strain of T. harzianum found on the fresh marine sponge Micale cecilia [103]. Cultivation of the strain in either fresh water medium or salty water medium gave significantly different profiles of metabolites. Extraction of the salty water medium yielded 83. This compound is probably an octaketide which has undergone a putative Diels-Alder reaction (Scheme 13) similar to that presumed to occur in the biosynthesis of lovastatin, a... 

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