Activities of amino acid metabolizing enzymes in the stomach and small intestine of developing rats

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A dose-response study of the bioavailability of grape seed proanthocyanidin in rat and lipid-lowering effects of generated metabolites in HepG2 cells

10.1016/j.foodres.2014.07.019Hyperlipidemia is one of the principal causes of cardiovascular disease and proanthocyanidins (PAs) regulate lipid homeostasis. This study aims to evaluate the concentration of PAs in rat serum after the administration of different doses of PAs and to determine the capacity of these metabolites to reduce de novolipid synthesis in HepG2 cells. Two hours after oral administration of different doses of a grape seed proanthocyanidin extract (GSPE) (1000, 375, 250 and 125 mg/kg), serum was semi-purified and characterised by HPLC¿ESI¿MS/MS before analysing the synthesis and secretion of lipids in HepG2 cells. Results showed a dose-dependent appearance of metabolised PAs in serum at doses up to 375 mg/kg and saturation at 1000 mg/kg of GSPE. A reduction in cholesterol esters (CE), free cholesterol (FC) and triglycerides (TG) synthesis was observed without dose-dependence when the cells were treated with PAs metabolites. Moreover, a low dose of metabolites (125 mg/kg) was sufficient to reduce FC and TG synthesis. In conclusion, the study demonstrated that PAs metabolise in a dose-dependent manner up to 370 mg/kg but not dose-dependent effect was shown in reducing the de novosynthesis of lipids

Assessment of Compatibility between Extraction Methods for NMR- and LC/MS-Based Metabolomics

10.1021/ac3005567Because of the wide range of chemically and structurally diverse metabolites, efforts to survey the complete metabolome rely on the implementation of multiplatform approaches based on nuclear magnetic resonance (NMR) and mass spectrometry (MS). Sample preparation disparities between NMR and MS, however, may limit the analysis of the same samples by both platforms. Specifically, deuterated solvents used in NMR strategies can complicate LC/MS analysis as a result of potential mass shifts, whereas acidic solutions typically used in LC/MS methods to enhance ionization of metabolites can severely affect reproducibility of NMR measurements. These intrinsically different sample preparation requirements result in the application of different procedures for metabolite extraction, which involve additional sample and unwanted variability. To address this issue, we investigated 12 extraction protocols in liver tissue involving different aqueous/organic solvents and temperatures that may satisfy the requirements for both NMR and LC/MS simultaneously. We found that deuterium exchange did not affect LC/MS results, enabling the measurement of metabolites by NMR and, subsequently, the direct analysis of the same samples by using LC/MS with no need for solvent exchange. Moreover, our results show that the choice of solvents rather than the temperature determined the extraction efficiencies of metabolites, a combination of methanol/chloroform/water and methanol/water being the extraction methods that best complement NMR and LC/MS analysis for metabolomic studie