| Metabonomics has been increasingly highlighted in "omics" technology, which is to study the systematic changes of all the metabolites in response to external stimuli. Metabonomics is the extension of genomics and proteomics, reflecting the biological event that has occurred. Metabonomics research, in addition to the changes of drug metabolism themselves, centers on the drug-induced changes of endogenous metabolites, which can shed light on the process of diseases comprehensively and offer new ideas about disease prevention and diagnosis.Currently, metabonomics has been widely used in toxicology, genomics, clinical diagnosis, clinical treatment, assessment of treatment, nutrition and molecular epidemiology, most of which are still in the preliminary research stage, especially in the analytical method. How to establish a rational method is still an urgent problem to be solved.A metabonomic method, based on GC/TOF-MS of endogenous metabolites in small rat serum, as well as on the appropriate data processing, pattern recognition and statistical analysis methods, has been successfully applied to D-GalN/LPS-induced mouse model of fulminant hepatic failure(FHF) and facilitate the search for metabolic markers that, if linked to substance pathways, may also be used for early prognosis of FHF.Firstly, a metabonomic method was established based on GC/TOF-MS of endogenous metabolites in plasma of mice.plasma metabolite extraction conditions based on relevant literatures were developed, The extraction efficiency of different organic solvents, singly and in combination, was investigated to optimize the extraction condition of endogenous metabolites.The result of the method,500μL of methanol,20μL of ribitol,450μL of pure water and 250μL of chloroform were added in Plasma samples(100μL) simultaneously, showed that its number and intensity of peak surpassed those of other methods By relevant statistical analysis.On the basis of the mass spectral database(NIST05) and AMDIS software,269 peaks were detected in plasma samples, the similarity of 48 of which was above 7000 and confirmed to be endogenous metabolites in the TIC detected by GC/TOF-MS.Eighteen endogenous metabolites were selected to investigate the system stability based on GC/TOF-MS technology at random from forty-eight metabolites.The result indicated most of the metabolites demonstrated good reproducibility, with intra-day, inter-day and stored in 4℃precision values below 15%. The relevant peaks were confirmed by standard compounds of monosaccharides and amino acids. The metabolites libraries of plasma were thus established, including 38 metabolites in the plasma library. These database can facilitate increasing the speed and accuracy of the peaks'identification in TIC.Secondly, Metabonomic Analysis for D-GalN/LPS-induced mouse model of Fulminant Hepatic Failure(FHF).12 male BALB/c mice were housed in a standard animal laboratory with a 12 h light-dark cycle and were provided with water and standard mouse chow ad libitum. The animals were randomly divided into GalN/LPS-induced FHF(n=6) and control(n=6)groups.On the basis of the mass spectral database,267 peaks were detected,52 of which were confirmed to be endogenous metabolites, such as sugars, amino acids, fatty acids, and organic acids, among other compounds. Of the 52 metabolites,48 were identified, whereas the others were unidentified metabolites in which significant differences were noted between the FHF group and the controls. These substances have been implicated in multiple biochemical processes, including energy and substance metabolism.Marked changes in plasma amino acids were observed on GC/TOF-MS chromatograms following GalN/LPS treatment. Though 16 amino acids were identified in total, amino acid concentrations were generally elevated in the FHF group, with significant differences in the levels of 15 amino acids(alanine,valine, isoleucine, glycine, serine, threonine, proline, leucine,aspartate, phenylalanine, glutamine, glutamate, ornithine,lysine, and tyrosine), with the exception of tryptophan, in which the increase was not statistically significant. The concentrations of total amino acids and gluconeogenic amino acids also increased. Furthermore, the ratio of BCAA to AAA (valine+leucine+isoleucine)/(tyrosine+phenylalanine+tryptophan) was significantly reduced in the FHF group.Compared with the control group,β-hydroxybutyrate(HB), fumarate, aminomalonic acid, malate and uric acid were elevated in the treatment group. Interestingly, levels of enolpyruvate(EP), succinate, and tetradecanoic acid decreased significantly among the nonamino-organic acids identified in FHF mice. Although there was a noted decrease in galactose and D-altrose in these mice, no significant differences in other carbohydrates, including glucose, were noted. Elevated phosphate,γ-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT), and 5-hydroxyindol-eaceti acid(5-HIAA)levels were also observed after GalN/LPS treatment. Levels of urea, palmic acid and stearic acid were similar in both groups.based on changes in the levels of intermediates in the FHF mice and the mechanisms underlying GalN/LPS damage to the liver, the pathways included gluconeogenesis, glycolysis, production of ketone bodies, the tricarboxylic acid(TCA) and urea cycles, among others. Whereas no significant differences between the fasted control and treated mice were noted in the gluconeogenic and glycolytic pathways, the production of ketone bodies, the TCA cycle, and the urea cycle were inhibited in the FHF mice. Although the levels of almost all amino acids were elevated in these mice, only a minority(e.g., aspartate, phenylalanine, and tyrosine) contributed to gluconeogenesis.Interpretation of the metabolite level data is difficult, not only because biochemical pathways are linked and highly regulated but also because information is lost in the process of averaging. Analysis methods, such as PCA, reduce data complexity by focusing on the information content of a given data set, while maintaining most of the information in a few dimensions. Analysis with SIMCA-P and PCA softwares indicated distinct clustering was observed between the FHF group and the control group.5-HIAA, glucose, HB, and phosphate are the parameters with the highest weights, and thus they had the greatest impact on each of the principal components in each experimental group.In all, based on GC/TOF/MS metabonomic method was establishped and applied for GalN/LPS-induced FHF. By the analysis of the metabolic profiling of plasma, significant differences in the plasma levels of many metabolites were noted, compared with those of the controls in the model of GalN/LPS-induced FHF. Furthermore, the earliest metabolic perturbations detected included the inhibition of ketogenesis and the TCA and urea cycles, with no significant changes in the gluconeogenic and glycolic pathways. PCA data analysis suggests that a combination of 5-HIAA, glucose, HB, and phosphate concentrations in the plasma is a potential marker for FHF. The above results demonstrate that this metabonomic approach is a powerful tool with which to characterize changes in the metabolic level, and to facilitate the search for metabolic markers under certain physiopathological conditions, which may be used for early prognosis of FHF. |
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