Gas chromatography - mass spectrometry based metabolomics study in OGD-induced SH-SY5Y cells following human mesenchymal stem cell treatment

Alternative Title
Alternative Author(s)
Gwang Lee
일반대학원 분자과학기술학과
The Graduate School, Ajou University
Publication Year
Gas chromatography mass spectrometryMetabolomicsMesenchymal stem cellOrganic acidAmino acidFatty acidPolyamine
Alternative Abstract
Gas chromatography - mass spectrometry (GC-MS) based metabolomics approach appears to be the method of choice for metabolic profiling analysis for monitoring of particular physiological or developmental state. Human mesenchymal stem cells (hMSC) have been known to enhance the viability and may have capability for restoration of metabolic function. hMSCs are also expected to possess cytoprotective and proliferative effects onoxygen glucose deprivation (OGD) exposed cells. Therefore, evaluation of the effect of oxygen tension and glucose concentration on stem cell biology, as well as the therapeutic potential of hMSCs on metabolic regulation thus has become necessary. In this study, GC-MS was used for analysis of metabolicprofilesand identifying biomarkers in neuroblastoma SH-SY5Y cells and culture media under OGD following hMSC treatment. Three derivatization methods were developed and optimized for the simultaneous measurement of 17 keto acids by GC-MS in selected ion monitoring (SIM) mode. Carbonyl groups present in keto acids in aqueous phase were directly converted into either ethoxime (EOX), tert-butyloxime (BuOX) or benzyloxime (BeOX) derivatives. After extraction and evaporation, tert-butyldimethylsilylation (TBDMS) of remaining carboxyl groups was performed. The electron impact mass spectral data and structures of 17 keto acids as EOX/TBDMS, BuOX/TBDMS and BeOX/TBDMS derivatives were newly established and confirmed. A variety of parameters that can affect the chemical EOX/TBDMS derivatization process such as pH condition, reagent, reaction time and temperature had been identified and optimized. Under the optimized conditions, all three methods for analysis of keto acids were fully validated with good linearity, precision and accuracy that indicated the suitability of these methods for analysis of metabolic profiles in biological samples. The present methods were successfully applied to measure the levels of organic acids in rat plasma and brain tissues. For analysis of metabolic profiles in cells and media samples, amino acids, organic acids, fatty acids and polyamines were converted into their ethoxycarbonyl/tert-butyldimethylsilyl (EOC/TBDMS), methoxime (MOX)/TBDMS, TBDMS and EOC/pentafluoropropionyl (PFP) derivatives, respectively, prior to be determined by GC-MS. All methods for assay of these metabolites were also validated. The analytical parameters obtained from validation were acceptable that indicate the adequate applicability of these methods for cell and media samples. These methods were successfully applied for profiling analysis of almost important intermediates of central pathways in cellular metabolism. The levels of total of 93 metabolites including 29 amino acids, 14 organic acids, 17 keto acids, 24 fatty acids and 9 polyamines were determined in cells and culture media samples from three groups: control, OGD and OGD co-cultured with hMSCs (OGD+hMSC), after cultivation for 24h and 72h. The disturbance in the levels of metabolites from cell and media of each group was demonstrated for studying effects of OGD condition and evaluation capability of hMSCs to metabolic function. Compared to the control group, significant changes of metabolite levels in cells and media from the OGD and OGD+hMSC groups were observed. The alteration in the levels of organic acids, amino acid, fatty acids and polyamines indicated the dysfunction of metabolic pathways in cells due to deficiency of oxygen and glucose. These changes may be associated with energy metabolism in cell during developmental stages. Therefore, the finding provided more obvious evidences for understanding insight biochemical reactions and explanation of hMSCs ability. The differences between the OGD and OGD+hMSC groups were also investigated. The levels of metabolites from SH-SY5Y cells which were co-cultured with hMSCs had a tendency to return to the range of the control values and restore to normal state. These finding suggested the potential effect of hMSCs in the restoration of cell metabolism and functions, and recovery of physiological levels of metabolites in cells. According to the significant P values obtained from Student’s t-test, some metabolites were suggested as potential biomarkers for monitoring therapeutic effects of hMSCs. To the best of my knowledge, this is the first comprehensive demonstration of changes in levels of a greatnumber of major mammalian metabolites including organic acids, amino acids, fatty acids and polyamines in the OGD-induced SH-SY5Y cells following human mesenchymal stem cell treatment. These finding indicated the vital role of oxygen and glucose in cell viability and development, which causes the changes in energy metabolism and alters cellular metabolite levels. The results also suggested that hMSCs canpotentially restore metabolic functions and promote physiological recovery in OGD-induced cell model, which provided additional evidences to prove the ability of hMSCs as a promising cell therapy approach in the future.

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Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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