검색 상세

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

초록/요약

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.

more

목차

DISSERTATION DECLARATION i
ACKNOWLEDGEMENTS ii
ABSTRACT iii
TABLE OF CONTENTS vi
LIST OF FIGURES xi
LIST OF TABLES xiii
ABBREVIATIONS xv
1. INTRODUCTION 1
1.1. Mesenchymal stem cell 1
1.2. Energy metabolism in cell 6
1.2.1. Glycolysis 7
1.2.2. Pentose phosphate pathway 9
1.2.3. Tricarboxylic acid cycle 9
1.2.4. Fatty acids degradation 10
1.2.5. Amino acids metabolism 11
1.3. Metabolism in human health and disease 13
1.3.1. Organic acid abnormalities 13
1.3.2. Amino acid abnormalities 18
1.4. Oxygen glucose deprivation 21
1.5. Metabolomics approach 23
1.6. Derivatization techniques in GC and GC-MS analysis 26
1.6.1. Silylation 27
1.6.2. Acylation 28
1.6.3. Alkylation/esterification 29
1.6.4. Alkyloxycarbonylation 31
1.6.5. Oximation 31
1.6.6. Combination methods 34
1.7. Aims of this study 35
2. EXPERIMENTAL 37
2.1. Chemicals and reagents 37
2.2. Preparation of standard solutions 42
2.3. Gas chromatography-mass spectrometry 42
2.4. Derivatization procedure 44
2.4.1. Sequential methoximation/tert-butyldimethylsilylation for analysis of organic acids including keto acids 44
2.4.2. A variety of sequential alkyloximation combine with tert-butyldimethylsilylation for analysis of keto acids 44
2.4.3. tert-Butyldimethylsilylation for analysis of fatty acids 44
2.4.4. Sequential ethoxycarbonylation/tert-butyldimethylsilylation for analysis of amino acids 49
2.4.5. Sequential ethoxycarbonylation/pentafluoropropionylation for analysis of polyamines 49
2.5. Method optimization for analysis of keto acids as EOX/TBDMS derivatives 52
2.6. Method validation for assay of amino acids, fatty acids, organic acids and polyamines 52
2.7. SH-SY5Y cells and human MSC culture 53
2.8. Rat plasma and brain tissue samples under transient MCAO model 54
2.9. Sample preparation for assay of organic acids, amino acids, fatty acids and polyamines in cells and culture media 54
2.10. Sample preparation for assay of organic acids in plasma and brain tissues from rat 55
2.11. Star symbol plotting 55
3. RESULTS AND DISCUSSION 56
3.1. Method development for analysis of keto acids 56
3.1.1. Ethoximation combined with tert-butyldimethylsilylation 56
3.1.2. tert-butyloximation combined with tert-butyldimethylsilylation 62
3.1.3. Benzyloximation combined with tert-butyldimethylsilylation 67
3.2. Method optimization for analysis of keto acids as EOX/TBDMS derivatives 72
3.2.1. Effect of pH condition 72
3.2.2. Effect of concentration of ethoxyamine hydrochloride and TDPA 74
3.2.3. Effect of reaction time and temperature 74
3.2.4. Stability of the EOX/TBDMS derivatives 78
3.3. Method validation for profiling analysis of keto acids 78
3.3.1.Method validation for assay of keto acids as EOX/TBDMS 78
3.3.2. Method validation for assay of keto acids as BuOX/TBDMS 81
3.3.3. Method validation for assay of keto acids as BeOX/TBDMS 84
3.4. Profiling analysis of organic acids in rat brain tissues 87
3.4.1. Method validation for profiling analysis of organic acids as EOX/TBDMS derivatives 87
3.4.2. Levels of organic acids in rat brain tissues 91
3.5. Profiling analysis of organic acids in cells and media 96
3.5.1. Method validation for profiling analysis of organic acids as MOX/TBDMS derivatives 96
3.5.2. Levels of organic acids in cells 100
3.5.3. Levels of organic acids in media 110
3.6. Profiling analysis of amino acids in cells and media 117
3.6.1. Method validation for profiling analysis of amino acids 117
3.6.2. Levels of amino acids in cells 121
3.6.3. Levels of amino acids in media 128
3.7. Profiling analysis of fatty acids in cells and media 135
3.7.1. Method validation for profiling analysis of fatty acids 135
3.7.2. Levels of fatty acids in cells 139
3.7.3. Levels of fatty acids in media 146
3.8. Profiling analysis of polyamines in cells and media 152
3.8.1. Method validation for profiling analysis of polyamines 152
3.8.2. Levels of polyamines in cells 156
3.8.3. Levels of polyamines in media 164
4. CONCLUSIONS 170
4.1. Summary 170
4.2. Further studies 173
REFERENCES 175
Appendix 1: GC-MS-SIM methods 185
A.1.1. GC-MS-SIM method for analysis of FAs as mono-TBDMS derivatives 185
A.1.2. GC-MS-SIM method for analysis of AAs as EOC/TBDMS derivatives 186
A.1.3. GC-MS-SIM method for analysis of PAs as EOC/PFP derivatives 187
A.1.4. GC-MS-SIM method for analysis of KAs as MOX/TBDMS derivatives 188
A.1.5. GC-MS-SIM method for analysis of OAs as MOX/TBDMS derivatives 189
A.1.6. GC-MS-SIM method for analysis of KAs as EOX/TBDMS derivatives 190
A.1.7. GC-MS-SIM method for analysis of OAs as EOX/TBDMS derivatives 191
A.1.8. GC-MS-SIM method for analysis of KAs as BuOX/TBDMS derivatives 192
A.1.9. GC-MS-SIM method for analysis of KAs as BeOX/TBDMS derivatives 193
Appendix 2: Electron-impact mass spectra of keto acids as EOC/TBDMS derivatives 194
Appendix 3: Electron-impact mass spectra of keto acids as BuOC/TBDMS derivatives 202
Appendix 4: Electron-impact mass spectra of keto acids as BeOC/TBDMS derivatives 208

more
반출 Meta View 목록