METABOLOMIC STUDIES OF THE ACUTE INFLAMMATORY RESPONSE TO SEVERE TRAUMA ...

311 Pages·2016·7.58 MB·English

METABOLOMIC STUDIES OF THE ACUTE
INFLAMMATORY RESPONSE TO SEVERE
TRAUMA INJURIES

ANNA KAREN CARRASCO LASERNA
(B.Sc., University of the Philippines Los Baños)

A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE

2015
Declaration

I hereby declare that this thesis is my original work and it has been written by
me in its entirety under the supervision of Professor Li Fong Yau, Sam (in the
S5-02-04/05 laboratories), Chemistry Department, National University of
Singapore, between 03 August 2009 and 27 March 2015.
I have duly acknowledged all the sources of information which have been used
in the thesis.
This thesis has also not been submitted for any degree in any university
previously.
The content of the thesis has been partly published in:
Not applicable, manuscripts in preparation

Anna Karen Carrasco Laserna 30 March 2015
Name Signature Date
Acknowledgement
Acknowledgement
I would like to express my deepest gratitude to Prof. Sam Li for
generously supporting and guiding me throughout the course of my PhD
studies. For sharing his wisdom and knowledge, for his patience, and for
giving me immense opportunities to learn, I am truly grateful. I would also
like to express my sincere gratitude to my co-supervisor, A/Prof. Shabbir M.
Moochala, for giving me the opportunity to work on these collaboration
projects, for his patience and for sharing his expertise.
I would also like to extend my sincere gratitude to NUS for providing
me the scholarship to pursue my PhD studies. I would like to thank the
Chemistry Department staff, especially Ms. Suriawati Binte Saad and Madam
Agnes Ang of the Admin office, and to Madam Han Yanhui and Dr. Wu Ji’en
of the CMMAC NMR facilities for their kind assistance throughout my
studies. I would also like to thank Prof. Loh Kian Ping, Prof. Yu Hao, Ms.
Suzana Binte Sulaiman, and Ms. Teo Chwee Hoon for their administrative
support and consideration.
Many thanks as well to the DMERI-DSO staff- Dr. Rajaseger
Ganapathy, Dr. Jor Ming, Dr. Wong Yong Chiat, Dr. Lai Yiyang, Dr. Wu Jian,
A/Prof. Mahesh Uttamchandani and Mr. Pavandip Singh, for providing the
samples and protein markers data, as well as for their valuable inputs. I would
also like to thank the doctors and staff at the NUH Department of Surgery,
especially to A/Prof. Dr. Philip Iau Tsau, Chief of General Surgery, and Ms.
Mona Hum Poh Lin, for the sample collection and provision of the clinical
data for the trauma patients.
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Acknowledgement
My sincere gratitude also goes to the staff at NERI, which has been my
second home in NUS, especially to Ms. Elaine Tay, Ms. Frances Lim, and Ms.
Per Poh Geok for their generous support, words of encouragement, and for all
the extra help. I would also like to thank the application specialists and service
engineers of AB Sciex, Dionex (Thermo Fisher), Agilent, and Scientific
Resources for their technical support with the instruments. Many thanks as
well to Dr. Etienne Thevenot of Metabohub, for introducing me to the use of
Cytoscape for network analysis.
I would also like to thank my colleagues in Prof Sam Li’s group for their
support and words of encouragement. Special thanks to my seniors: Dr. Lau
Hiu Fung and Dr. Fang Guihua, for their patience and for generously sharing
with me their knowledge as I was starting my studies. Special thanks as well
to my NERI lab mates and friends, Zhang Wenlin and Lee Si Ni, for all their
help and support as I was completing my experiments and preparing this
dissertation, as well as for the constantly fun and insightful discussions. I
would like to thank my previous UROPS, honours and MSc students, Koh
Xue Qi, Shawn Foo Siong Chun, Ang Xiu Qin, Lim Wanrong, Erhan Simsek,
and Hamza Yuksel, for giving me the privilege to pay forward by allowing me
to share my knowledge and learnings through the projects that we have
worked in together.
Thank you to all of my friends, former colleagues and superiors, as well
as to my brothers and sisters in my church ministries, for their words of
encouragement and prayers. To my Breaking Pandesal family- Helen,
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Acknowledgement
Stephany, Gian and Emmanuel, thank you so much for the constant prayers &
support.
My love and deepest gratitude goes to my family, especially to my
parents- for their unwavering love and support, for always believing in me,
and for all the sacrifices. I love you all and thank you so much.
To my Lord and God, from whom all good things come, I thank you
with all my heart for this challenging yet truly grace-filled chapter of my life.
Thank you for allowing me to encounter you in this journey and I offer all this
up to you in thanksgiving. All glory and honour is yours forever.
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Table of Contents
Table of Contents
Acknowledgement .............................................................................................. i
Table of Contents .............................................................................................. iv
Summary ........................................................................................................... ix
List of Tables .................................................................................................... xi
List of Figures .................................................................................................. xii
List of Abbreviations ...................................................................................... xiv
List of Symbols ................................................................................................ xx
Chapter 1. Introduction ..................................................................................... 1
1.1. Severe injuries and the inflammatory response ........................................ 1
1.1.1. Epidemiology of injuries .............................................................. 1
1.1.2. The acute inflammatory response and its complications .............. 2
1.2. Metabolomics ........................................................................................... 9
1.2.1. Metabolomics approach to biomarker discovery ........................ 10
1.2.2. Workflow of metabolomic studies ............................................. 11
1.2.2.1. Sample collection and preparation ............................... 14
1.2.2.2. Analytical platforms used in metabolomics studies ..... 18
1.2.2.3. Processing of metabolomics data ................................. 32
1.2.2.4. Statistical analyses used in metabolomic studies ......... 36
1.2.2.5. Metabolite identification .............................................. 40
1.2.2.6. Metabolic pathway and metabolic network analysis .... 42
1.3. Metabolomics in the study of the inflammatory response in severe
injuries: a systems approach to a systemic problem ............................... 43
1.4. Scope of Work ........................................................................................ 49
1.5. References .............................................................................................. 50
Chapter 2. Comprehensive metabolomic profiling of simulated combat
trauma injury in a porcine model using NMR and LC-MS .................... 94
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Table of Contents
2.1. Introduction ............................................................................................ 94
2.2. Experimental Procedure ......................................................................... 99
2.2.1 Chemical Reagents ..................................................................... 99
2.2.2 Animal Experiment ..................................................................... 99
2.2.2.1 Anaesthesia ................................................................. 100
2.2.2.2 Instrumentation ........................................................... 100
2.2.2.3 Injury Phase ................................................................ 101
2.2.3 Sample Preparation ................................................................... 103
2.2.3.1 Sample Preparation for NMR Analysis ...................... 103
2.2.3.2 Sample Preparation for LC-MS Analysis ................... 103
2.2.4 Instrumental Analysis ............................................................... 104
2.2.4.1 NMR Analysis ............................................................ 104
2.2.4.2 Liquid Chromatography ............................................. 104
2.2.4.3 Mass Spectrometry ..................................................... 105
2.2.5 Data Processing and Analysis ................................................... 106
2.2.5.1 Data Pre-processing for NMR .................................... 106
2.2.5.2 Data Pre-processing for LC-MS ................................. 106
2.2.5.3 Data Analysis.............................................................. 106
2.3. Results and Discussion ......................................................................... 108
2.3.1. 1H NMR Data Analysis ............................................................ 108
2.3.2. LC-MS Analysis ....................................................................... 116
2.3.3. Metabolic Pathways Analysis ................................................... 124
2.4. Conclusion ............................................................................................ 134
2.5. References ............................................................................................ 135
Chapter 3. Correlation network analysis reveals insights into the acute
inflammatory response and organ damage in a porcine model of combat
trauma injury ........................................................................................ 148
3.1. Introduction .......................................................................................... 148
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Table of Contents
3.2. Experimental Procedure ....................................................................... 150
3.2.1. Animal Experiment ................................................................... 150
3.2.2. Blood Sampling and Protein Markers Analyses ....................... 151
3.2.3. Data Analysis ............................................................................ 152
3.3. Results and Discussion ......................................................................... 153
3.3.1. Inflammatory and organ damage markers ................................ 153
3.3.2. Correlation analysis .................................................................. 158
3.3.3. Identification of potential organ-specific injury markers ......... 161
3.3.4. Correlation network analysis .................................................... 166
3.3.4.1. Myocardial injury sub-network .................................. 168
3.3.4.2. Renal injury sub-network ........................................... 174
3.3.4.3. Cysteine-methionine metabolism sub-network .......... 180
3.4. Conclusion ............................................................................................ 183
3.5. References ............................................................................................ 184
Chapter 4. Metabolomic study of the development of multiple organ
dysfunction syndrome (MODS) in trauma patients.............................. 205
4.1. Introduction .......................................................................................... 205
4.2. Experimental procedure........................................................................ 207
4.2.1. Patient recruitment .................................................................... 207
4.2.2. SOFA Assessment .................................................................... 209
4.2.3. Cytokine, protein markers and immunomodulatory gases
(CPMG) ................................................................................................ 210
4.2.4. Metabolomics analysis ............................................................. 210
4.2.4.1. Standards and reagents ............................................... 210
4.2.4.2. Sample preparation ..................................................... 211
4.2.4.3. Liquid chromatography .............................................. 211
4.2.4.4. Mass spectrometry ...................................................... 212
4.2.4.5. Data pre-processing .................................................... 212
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Table of Contents
4.2.4.6. Data analysis ............................................................... 213
4.3. Results and discussion .......................................................................... 214
4.3.1. Patient characteristics & SOFA scores ..................................... 214
4.3.2. Quality control samples ............................................................ 216
4.3.3. OPLS-DA analysis of the datasets ............................................ 217
4.3.4. Identification of significant metabolic features ........................ 219
4.3.5. Metabolic pathway analysis ...................................................... 226
4.3.6. Inflammatory and organ damage protein markers .................... 227
4.3.7. Correlation with organ system and total SOFA scores ............. 229
4.4. Conclusion ............................................................................................ 234
4.5. References ............................................................................................ 235
Chapter 5. Conclusion and future work ........................................................ 247
Appendices ..................................................................................................... 252
Appendix 1. Fold-change comparison of significant features from NMR
binned data (p<0.05). ................................................................................ 252
Appendix 2. Comparison of profiled metabolite concentrations using
Chenomx Profiler. ..................................................................................... 255
Appendix 3. Fold change values of LC-MS metabolites identified to be
significantly changed after trauma. ........................................................... 257
Appendix 4. Comparison of base peak chromatograms of the HILIC LC-
MS analyses for the sham and trauma samples. ........................................ 264
Appendix 5. Comparison of base peak chromatograms of the RPLC-MS
analyses for the sham and trauma samples. .............................................. 265
Appendix 6. Shewhart control charts of the LC-MS pooled QC samples.266
Appendix 7. Comparison of the cytokines and other clinical markers for
sham and injured pigs. ............................................................................... 267
Appendix 8. Abbreviations of compound names used in network analysis.
................................................................................................................... 269
Appendix 9. Shewhart control charts for the pooled QCs of the trauma
patient samples. ......................................................................................... 272
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Table of Contents
Appendix 10. OPLS-DA score plots for MODS and non-MODS
differentiation using different thresholds for the different datasets. ......... 273
Appendix 11. List of metabolites differentiating MODS and non-MODS
samples. ..................................................................................................... 274
Permissions for Reproduced Figures ............................................................. 279
Presentations and Manuscripts in Preparation ............................................... 289

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