Use of metabolomics in biomedical and environmental studies

USE OF METABOLOMICS IN BIOMEDICAL AND ENVIRONMENTAL STUDIES HUANG SHAOMIN B.SC. (HONS), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY SAW SWEE HOCK SCHOOL OF PUBLIC HEALTH NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. 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 Huang Shaomin A0031110M 13 August 2014 i ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my PhD supervisor, Professor Ong Choon Nam (Saw Swee Hock School of Public Health) for his invaluable mentorship and encouragement over the past years. The multidisciplinary training in the OCN laboratory has not broaden my horizons and expertise, but most importantly made my PhD studies both fulfilling and enriching. I would also like to acknowledge the NUS Research Scholarship, which provided strong research and educational support for my PhD education. I gratefully thank my collaborators for their guidance and support through my projects. To my seniors and friends in the OCN lab (Xu Fengguo, Xu Yongjiang, Gao Liang, Cui Liang, Jinling, Su Jin, Zou Li, Yonghai), I sincerely thank you for being truly great and generous people. Your patience and kindness have eased me well into learning more about metabolomics and mass spectrometry. I would further like to thank Dr Tan Chuen Seng for guiding me with his wealth of statistical and programming knowledge. I also thank Dorothy, Bee Lan, Mr Ong Her Yam and Ai Li for their guidance and support. My PhD education would not be complete without great friends and hence to Eugene & Wei Zhong, it has been my greatest pleasure to have known you. Both of you have greatly enriched my life perspective and education in NUS. Lastly, I would like to thank my family and friends, especially my mum and girlfriend. Their constant care and support keeps me persevering and striving for excellence and mastery. ii TABLE OF CONTENTS DECLARATION ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF PUBLICATIONS CONFERENCE ABSTRACTS SUMMARY LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS i ii iii viii ix x xvi xvii xx Chapter – Introduction of Metabolomics 1.1 Introduction .2 1.1.1 Metabolomics as a tool for understanding responses in biological systems .2 1.1.2 The role of metabolism and its implication in biological responses .5 1.1.2.1 Metabolism provides free energy to carry out biological functions 1.1.2.2 Specific metabolic pathways produce specific metabolites 1.1.2.3 Differential metabolite levels are driven by enzyme regulation 1.1.2.4 Discovering perturbed pathways and potential biomarkers 10 1.2 The discovery process in metabolomics .11 1.2.1 Experimental design 11 1.2.1.1 Sample type and variability reduction .11 1.2.1.2 Sample collection .14 1.2.1.3 Sample preparation 15 1.2.1.4 Sample injection order .16 1.3.1 Analytical Instruments .16 1.3.1.1 LC-MS and GC-MS as key analytical instruments 17 1.3.1.2 Derivatization of metabolites for GC-MS 18 1.3.1.3 Ionization modes in LC-MS and GC-MS and its implications for data analysis 19 1.3.2 Data analysis in metabolomics 21 1.3.2.1 Pre-processing 22 1.3.2.2 Normalization of Peaks 23 iii 1.3.2.3 Multivariate analysis 26 1.3.2.4 Univariate Analysis 28 1.3.2.5 Peak shortlisting and identification 28 1.3.2.6 Biological Inference .30 1.4 Objective of thesis: the application of metabolomics to biomedical and environmental studies .30 Chapter – Toxicological evaluation of silica nanoparticles using an in vitro model 2.1 Introduction .39 2.2 Materials and Methods 40 2.2.1 SiO2NP synthesis .40 2.2.2 Cell culture 41 2.2.3 Treatment of MRC-5 with SiO2NP .41 2.2.4 Metabolite extraction and chemical derivatization 42 2.2.5 GC-MS and LC-MS .42 2.2.6 Spectral data analysis .44 2.2.7 MTS cell viability assay & cell area calculation .45 2.2.8 Confocal microscopy & TEM .45 2.2.9 TEM examination of SiO2NP treated cells and EDX analysis (Energydispersive X-ray Microanalysis) 46 2.2.10 TBARS assay .46 2.2.11 Statistical analysis 47 2.3 Results .47 2.3.1 SiO2NP synthesis .47 2.3.2 MRC-5 cell line assay 48 2.3.3 Metabolomics findings 49 2.3.4 Electron microscopy reveals uptake of SiO2NP in vacuoles .53 2.4 Discussion .56 2.5 Conclusion 58 2.6 Acknowledgements 58 iv Chapter – Use of Zebrafish Embryos and Metabolomics to Assess Water Quality 3.1 Introduction .63 3.2 Materials and methods 64 3.2.1 Collection procedure 64 3.2.2 Extraction 65 3.2.3 GC-MS and LC-MS analysis .66 3.2.4 Mass spectrometry data pretreatment, marker metabolites selection and identification .69 3.2.5 3.3 mRNA transcript matching with target metabolite 70 Results .71 3.3.1 Clustering of metabolomic data shows changes during embryogenesis .71 3.3.2 Hierarchical clustering analysis and identification of metabolites 74 3.3.3 Linking metabolite levels to gene expression levels .77 3.3.4 Linking proteomic data to metabolite levels .81 3.3.5 Proof of concept: Applying zebrafish metabolomics on embryos exposed to NDMA 82 3.4 Discussion .86 3.5 Conclusion 93 Chapter – An integrated LC- and GC-MS approach for investigating non-proteinuric chronic kidney disease 4.1 Introduction .101 4.2 Materials & Methods 103 4.2.1 Patients and urine samples .103 4.2.2 Definitions of non-proteinuria and low eGFR .103 4.2.3 Metabolomic analysis using GC-MS .104 4.2.4 Metabolomic analysis using LC-MS .105 4.2.5 Metabolomic data preprocessing .106 4.2.6 Statistical analysis 107 4.3 Results .108 v 4.3.1 Patient characteristics 108 4.3.2 GC-MS analyses 109 4.3.3 LC-MS analyses 114 4.4 Discussion .118 4.5 Conclusion 122 Acknowledgements 123 Contribution statement .123 Chapter – MetaboNexus – an interactive platform for integrated metabolomics analysis 5.1 Introduction .128 5.2 Methods .130 5.2.1 Overall Design .130 5.2.2 Method of use and file input 134 5.2.2.1 Input 1: Pre-processing with MetaboNexus .135 5.2.2.1 Input 2: Pre-processing with other softwares (e.g. MZmine) 136 5.2.3 Starting MetaboNexus .137 5.2.3.1 Data transformation & annotation .137 5.2.3.2 Principal Component Analysis (PCA) .138 5.2.3.3 Partial Least Squares-Discriminant Analysis (PLS-DA) .138 5.2.3.4 Random Forest (RF) 140 5.2.3.5 Merging Variable Importance with Univariate Analysis .140 5.2.3.6 Metabolite Search & Pathway Information .141 5.2.3.7 Heatmap .142 5.3 Results .143 5.3.1 Evaluating performance of MetaboNexus .143 5.3.2 User experience .145 5.4 Conclusion 147 Chapter – Conclusions, Limitations and Outlook vi 6.1 Conclusions .150 6.2 Limitations and Outlook .152 6.3 Metabolite identification .152 6.4 Biological Interpretation .153 6.5 Scalability of experiments .155 References 157 vii LIST OF PUBLICATIONS 1. Ng DP, Salim A, Liu Y, Zou L, Xu FG, Huang S, Leong H, Ong CN. A metabolomic study of low estimated GFR in non-proteinuric type diabetes mellitus. Diabetologia. 2012 Feb;55(2):499-508 2. Huang SM, Zuo X, Li JJ, Li SF, Bay BH, Ong CN. Metabolomics studies show dose-dependent toxicity induced by SiO(2) nanoparticles in MRC-5 human fetal lung fibroblasts. Advanced Healthcare Materials. 2012 Nov;1(6):779-84 3. Huang SM, Xu F, Lam SH, Gong Z, Ong CN. Metabolomics of developing zebrafish embryos using gas chromatography- and liquid chromatography-mass spectrometry. Molecular Biosystems. 2013 Jun;9(6):1372-80 4. Huang SM, Toh WZ, Benke PI, Tan CS, Ong CN. MetaboNexus: an interactive platform for integrated metabolomics analysis. Metabolomics 2014 Dec 10(6):108493 5. Gao Y, Lu Y, Huang SM, Gao L, Liang X, Wu Y, Wang J, Huang Q, Tang L, Wang G, Yang F, Hu S, Chen Z, Wang P, Jiang Q, Huang R, Xu Y, Yang X, Ong CN. Identifying Early Urinary Metabolic Changes with Long-Term Environmental Exposure to Cadmium by Mass-Spectrometry-Based Metabolomics. Environmental Science and Technology. 2014, May 48 (11), 6409-18 6. Ho WE, Xu YJ, Xu FG, Cheng C, Peh HY, Huang SM, Tannenbaum SR, Ong CN, Wong FWS. Anti-malarial drug artesunate restores metabolic changes in experimental allergic asthma. Metabolomics. 2014 July (e-publication) All publications have been reviewed by international referees. viii CONFERENCE PRESENTATIONS 1. Singapore Water Week 2012  “Use of zebrafish embryo for water quality assessment; an integrated genomic and metabolomics approach” 2. Lhasa Toxicity Symposium 2012 – New Horizons in Toxicity Prediction, Cambridge, United Kingdom  “Metabolomics as a tool for nanotoxicity assessment – a dual in vitro and in vivo approach” 3. 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Analytical and Bioanalytical Chemistry, 394(1), 47–59. 172 [...]... reproducible and accessible by collaborators and broader readership The overall findings of this thesis demonstrate that metabolomics can complement and add value to existing approaches in biomedical and environmental research by providing a comprehensive and sensitive means of detecting and classifying biological responses in sample types of increasing complexity The development of an integrated software... in designing a metabolomics experiment In metabolomics, experiments are carefully designed from the initial sampling process to the final injection sequence During the collection of each sample type, the process itself may introduce undesired variability in the form of batch effect or confounding factors The sample integrity and metabolic profile is preserved by quenching metabolism and refrigerating... Further investigations involving ultrastructural studies revealed uptake of nanosilica through dose-dependently increased vacuolization Here the feasibility of metabolomics for in vitro investigations was demonstrated and metabolomics further complemented existing methodologies for toxicological assessment In vivo animal models are valuable in demonstrating and extrapolating clinical relevance of exposure... from the use of different batches of living organisms or from different operating conditions of instrument/reagent Often, batch effect is detected when the data are found to not correlate with variables of interest, but instead with different batches of organisms, reagents and instrument operating conditions Hence batch effect introduces systematic variation that are of a confounding nature and could... understand metabolic perturbation caused by Nnitrosodimethylamine (NDMA), a potent carcinogen present in drinking water The embryos were exposed to increasing doses of NDMA (0, 0.1, 1, 10 µg/L) with the exposure sustained up to 48 hpf Morphological inspection of zebrafish embryos and xii mortality counts revealed no significant effects of NDMA up to the highest dose of 10 µg/L of NDMA Despite the lack of. .. proteins and metabolites (Sauer, Heinemann, & Zamboni, 2007) Often, the responses generate and/ or modify a broad array of molecular components that are interdependent and integrated with each other For example, a transcription factor would initiate the expression of a certain class of genes in response to a stressor and the translation of these genes may further regulate other protein functions in the... and gender These factors would therefore rank urine as the most complex and variable sample type to analyse from the technical aspect In Chapter 4 we applied metabolomics to study the feasibility of urine metabolomics owing to the benefits of non-invasive sampling and ease of collection from the study population In the diagnosis of renal insufficiency, urine samples from patients are traditionally analysed... understanding biological responses and biomarker discovery Furthermore, these studies reaffirm that metabolite levels are indeed a valuable source of information for understanding diseases and toxicological responses Metabolomics is a rapidly-evolving discipline of systems biology, with major advances in analytical chemistry and related bioinformatics However, the current state of metabolomics analysis lacks... focus on recruiting a near-homogenous pool of subjects to eliminate confounders They are typically qualified for inclusion by matching factors such as age, gender, health status and lifestyle habits before their samples are used in a metabolomics experiment On top of eliminating confounders, careful sample preparation and sample injection is needed to avoid introducing batch effect during data acquisition... collection In vitro and in vivo samples contain cells that are capable of metabolism and in order to ensure a more accurate measurement of metabolites, the metabolism within these live samples needs to be quenched Metabolism quenching can be achieved by the use of rapid cooling methods such as snap freezing in liquid nitrogen or by washing the samples with cold buffer solutions After quenching is performed, . and environmental research by providing a comprehensive and sensitive means of detecting and classifying biological responses in sample types of increasing complexity. The development of an integrated. technical aspect. In Chapter 4 we applied metabolomics to study the feasibility of urine metabolomics owing to the benefits of non-invasive sampling and ease of collection from the study population. In the. USE OF METABOLOMICS IN BIOMEDICAL AND ENVIRONMENTAL STUDIES HUANG SHAOMIN B.SC. (HONS), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY SAW SWEE HOCK SCHOOL OF PUBLIC HEALTH NATIONAL