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Endogenous metabolites testing is a new technology to study all metabolic pathways in organisms by studying the changes of metabolites after stimulation or disturbance, which is modeled on the research ideas of genomics, transcriptomics and proteomics. The study subjects of endogenous metabolites testing are mainly endogenous small molecules with a relative molecular weight of less than 1000. Compared with other analytical methods, endogenous metabolites testing can reflect the physiological and pathological state of the body system more truly, and provide an instantaneous and comprehensive snapshot for the ongoing biological process.
Due to the active enzyme system and rapid metabolic transformation in cells, sampling and sample preparation methods significantly affect the accuracy and repeatability of analysis results. Therefore, establishing analytical standards for sampling and sample preparation methods of endogenous metabolites testing is important. However, according to the research objects, purposes and analysis techniques, the pretreatment requirements of samples are also inconsistent, and there are no analytical standards so far. In the process of samples pretreatments, the regents used are mainly methanol, sodium chloride, perchloric acid, acetonitrile, isopropyl alcohol and others.
Generally, the steps of sample treatment can be divided into cell quenching and metabolite extraction.
Cell quenching: The purpose of cell quenching is to inactivate the enzymes in the cell and ensure that the metabolite contour is frozen. As for intracellular metabolites, due to the changes in the metabolic state of cells after sampling or leaving the culture environment, the types and contents of metabolites also change accordingly. In order to correctly reflect the true information of cell metabolites in the culture environment, cells need to be quenched immediately and intracellular reactions need to be terminated. The ideal quenching technique should ensure the rapid inactivation of enzymes while maintaining the integrity of cells. The cell quenching methods include liquid nitrogen freezing method, acid-base inactivation method, rapid filtration method, low temperature centrifugation method, cell scraping method, cold methanol method and others. Methanol, hydroxyethyl piperazine ethyl thiosulfonic acid, ammonium bicarbonate and sodium chloride are generally used in this step.
Metabolite extraction: Once the metabolic reaction is stopped by cell quenching, the metabolites need to be extracted from the cell. According to the levels of endogenous metabolites testing, the metabolite extraction methods and requirements are also different. For example, in target analysis, specific methods should be used to selectively extract the corresponding components. In contour analysis, it is necessary to extract as many metabolites as possible. At present, many researchers have systematically investigated a variety of metabolite extraction methods, but due to the different types of cell lines, culture conditions, medium composition and cell physiological conditions, the metabolite level is quite different. There is still no unified analytical standards so far. However, the following principles should be followed in general. First, the maximum number of metabolites are extracted from the cells with an appropriate recovery rate. Second, metabolites should not undergo any physical or chemical modification to minimize degradation. Third, it is necessary to prevent the leakage of metabolites. Fourth, the extraction method needs to be non-destructive. In this step, the reagents used include cold methanol, hot ethanol, perchloric acid, chloroform, acetonitrile, trichloroacetic acid, isopropyl alcohol and others.
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