목차

I. General introduction. 1
1. The important stages of lipidomics workflow. 1
2. Analytical tools in lipid profiling methods 2
3. Mass spectrometry application in lipidomics. 3
4. Sphingolipid metabolic pathway 6
5. Deoxysphingolipid metabolism. 7
II. Chapter 1 10
LC-MS/MS-method establishment for comparative profiling of sphingolipids and deoxysphingolipids in mouse tissues 10
1. Introduction 10
2. Materials and methods. 12
2.1. Chemicals 12
2.2. Sample preparation 12
2.3. Lipid extraction. 12
2.4. LC-MS/MS condition 14
2.5. Method validation. 14
2.6. Statistical analysis. 15
3. Results 15
4. Discussion 36
III. Chapter 2 42
Comparative cell cytotoxicity of sphingolipids and deoxysphingolipids 42
1. Introduction 42
2. Materials and methods. 43
2.1. Reagents 43
2.2 Methods. 43
3. Results. 44
4. Discussion. 47
IV. Chapter 3 50
Comparative metabolic rate of sphingolipids 50
1. Introduction. 50
2. Materials and methods 50
2.1. Reagents 51
2.2. Cell culture and in vitro metabolism of deoxysphingolipids 51
2.3. Preparation of standart solutions for i.v. injection 52
2.4. Comparative ex vivo metabolism of deoxysphingolipids and sphingolipids 52
2.5. In vivo comparative metabolism of sphingolipids 52
2.6. Lipid extraction. 52
2.7. LC-MS/MS conditions 53
2.8. Statistical analysis. 53
3. Results. 53
3.1. In vitro metabolism of atypical sphingolipids. 53
3.2. Comparative ex vivo metabolism of sphingolipids. 60
3.3. Comparative in vivo metabolism of sphingolipids. 66
4. Discussion. 76
V. Chapter 4. 80
Sphingolipid profiling in chronic kidney disease model 80
1. Introduction. 80
2. Materials and methods 82
2.1. Reagents 82
2.2. In vivo CKD induction and biospecimen collection. 82
2.3. Cell culture 82
2.4. Preparation of kidney samples 83
2.5. Determination of the plasma creatinine level. 83
2.6. Detection of tryptophan and kynurenine levels in plasma 83
2.7. Effects of 2,8-dihydroxyadenine on sphingolipids. 83
2.8. SPT activity assay. 84
2.9. CerS activity assay 85
2.10. aSMase activity assay 85
2.11. Lipid extraction. 86
2.12. LC-MS/MS conditions 87
2.13. Statistical analysis. 87
3. Results. 87
3.1. General data of an adenine-induced CKD rat model 87
3.2. Lipid alteration in plasma and kidney tissues. 89
3.3. Lipid alteration in HK-2 cells 93
3.4. SPT activity assay results 95
3.5. CerS activity results 96
3.6. aSMase activity results 101
4. Discussion. 102
VI. Chapter 5 107
Comparative study of (deoxy)sphingolipids in MC903 induced Atopic dermatitis disease model 107
1. Introduction. 107
2. Materials and methods 109
2.1. Reagents 109
2.2. MC 903 solution preparation. 109
2.3. Preparation of 1.2% Avertin solution for anesthesia 110
2.4. Atopic dermatitis induction. 110
2.5. Serum TSLP level assay 111
2.6. Histological examination. 112
2.7. SPT activity assay. 112
2.8. CerS activity assay 113
2.9. aSMase activity assay. 113
2.10. Lipid extraction. 113
2.11. LC-MS/MS analysis of ceramides 114
2.12. Statistical analysis. 114
3. Results. 114
3.1. Phenotypic and skin structure change in AD model. 114
3.2. Endogenous sphingolipid alteration in the MC-903 atopic dermatitis induced mice model. 116
3.3. SPT activity assay results. 122
3.4. CerS activity assay results. 123
3.5. aSMase activity assay results 125
4. Discussion. 126
VII. Conclusions. 129
VIII. Reference list. 131
Abstract in Korean 150
Acknowledgements 152