MSACL 2020 US Abstract

A Novel UPLC-MS Approach for Metabotyping Burn Injury

Dominic Friston1, Helen Laycock1, Istvan Nagy1, Elizabeth J. Want2*
1. Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Imperial College, London ; 2. 1 Section of Biomolecular Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College, London

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Elizabeth Want (Presenter) Imperial College London

Presenter Bio: Dr Elizabeth Want is a Senior Lecturer in Molecular Spectroscopy in the Department of Metabolism, Digestion and Reproduction. She joined Imperial College in 2006 after working as a postdoctoral researcher at the Scripps Research Institute in La Jolla, CA. Prior to that, Dr Want obtained her PhD from King’s College London. At Imperial College, she was initially a postdoctoral researcher for the Consortium for Metabonomic Toxicology (COMET) group before becoming a Lecturer in 2007 and Senior Lecturer in 2014. She is also the Director of the Imperial International Phenome Training Centre. Dr Want has >20 yrs of experience in mass spectrometry and chromatographic techniques and has spent the past 18 years working in the field of mass spectrometry-based metabolite profiling. Her research focuses primarily on the development and application of novel mass spectrometry (MS) based techniques for

Relevant Financial Disclosures (within past 24 months) Committee/Board/Advisory Board MSACL EU Scientific Committee, Reid Bioanalytical Committee Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Burn injury affects millions of people worldwide and is often devastating, with diminished quality of life for many sufferers. Burns can result in life-limiting chronic pain, often refractory to treatment. Burns are categorised according to injury depth (1st-3rd degree), and treatment is dependent on burn thickness. However, mechanisms behind burn injury are poorly understood, with little research into their molecular basis and subsequent pain. Although important to explore the local signalling environment, studies are often destructive and preclude collection of longitudinal temporal data. Microdialysis allows the in vivo collection of solutes primarily from the extracellular interstitium, ideal for the study of burn injury. Metabolomics offers an unbiased approach to the elucidation of metabolites involved in pathological events. Coupled with chromatography and mass spectrometry, it provides a sensitive platform for the detection of metabolic changes due to burn injury.
OBJECTIVES: Elucidation of altered metabolites due to burn injury, to enhance understanding of pathological processes and guide future therapeutic strategies.
METHODS: Subcutaneous microdialysis in a novel burn model was coupled with ultra performance liquid chromatography – mass spectrometry (UPLC-MS) analysis (ESI+/- modes). Microdialysis was conducted for 0.5 hrs pre-burn and 3 hours post-burn, collected in 0.5 hour fractions for time series analysis. An Acquity UPLC system (HSS T3 column) was connected to a Synapt G2-S Q-ToF (Waters). Cytokine data were collected in parallel using ELISA. Data were processed using XCMS software. Principal components analysis (PCA) was used for unsupervised multivariate comparison of burn and control sites. Partial least squares discriminant-analysis (PLS-DA) was applied to rank discriminatory metabolite features. Model robustness was affirmed using CV-ANOVA. UPLC-MS/MS analysis was performed for structural elucidation.
RESULTS: Hundreds of polar analytes and lipids were profiled from microdialysate over an optimised 12 minute reversed phase run, yielding a high number of burn-altered metabolites. PCA scores plots showed tight quality control (pooled) sample grouping, indicating acquisition stability. Clear metabolic differences were observed after burn injury; with two identified metabolites, niacinamide and uric acid potentially involved in the pathology. Significant cytokine alterations were observed in response to burn injury.
CONCLUSION: This study demonstrates the application of high throughput metabolomic profiling to samples collected both continuously and specifically from the burn site. Further understanding of metabolic changes induced by burn injury will help to guide future therapeutic intervention. This approach is equally applicable to the analysis of other tissues and pathological conditions, so may further improve our understanding of the metabolic changes underlying a wide variety of pathological processes.