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University of Southampton Research Repository University of Southampton Research Repository Copyright © and Moral Rights for this thesis and, where applicable, any accompanying data are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis and the accompanying data cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content of the thesis and accompanying research data (where applicable) must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holder/s. When referring to this thesis and any accompanying data, full bibliographic details must be given, e.g. Thesis: Author (Year of Submission) "Full thesis title", University of Southampton, name of the University Faculty or School or Department, PhD Thesis, pagination. Data: Author (Year) Title. URI [dataset] UNIVERSITY OF SOUTHAMPTON RSITY OF SOUTHAMPTON Faculty of Medicine FACULTY OF YOUR_FACULTY Clinical and Experimental Sciences Academic_Unit Volume X of Y Capturing the intra- and extracellular redox metabolome by mass spectrometry - Thesis_Title Significance of sample processing and methodology by by Your_Name Thomas Rupert Sutton Thesis for the degree of Thesis for the degree of Doctor of Philosophy September 2019 Month_Year UNIVERSITY OF SOUTHAMPTON ABSTRACT Faculty of Medicine Clinical and Experimental Sciences Thesis for the degree of Doctor of Philosophy Capturing the intra- and extracellular redox metabolome by mass spectrometry - Significance of sample processing and methodology Thomas Rupert Sutton Oxidative stress has recently become understood as a disruption of redox signalling and control, and is strongly associated with a wide number of physiological conditions and diseases. Redox metabolism plays a key role in the defence of cells against oxidative damage. Attempts to produce better outcomes in these conditions by correcting the redox imbalance with antioxidants have produced mixed results at best. Therefore, there is an increasing need to better understand redox metabolism and how the many redox pathways involved are connected. In particular, hydrogen sulfide (H2S) has emerged as a key signalling molecule in redox regulation, along with its associated metabolites and redox pathways. Whilst there are existing analytical methods available for the analysis of H2S and its metabolites, they are often limited in scope, and neglect detailed investigation of important steps in the analysis including sample processing. The ascorbate metabolome comprises a separate redox pathway and has close links to the sulfide metabolome. Some aspects of the ascorbate metabolome, such as ascorbic acid-2-sulfate, however have not been widely analysed in humans. In order to better understand the sulfide redox metabolome, an ultra-high pressure liquid chromatography mass spectrometry (UHPLC-MS) based method was developed to quantify sulfide and other key metabolites. Important aspects of sample processing including collection, preparation and storage, were investigated to determine their impact on the measured metabolites. N-Ethylmaleimide was used to trap the sulfide and prevent oxidation and degradation of the other metabolites. The same method was then utilised to analyse persulfides and polysulfides, which are additional redox relevant sulfide related metabolites. The kinetics of the trapping reactions were also investigated in more detail. Additionally, a UHPLC-MS method for the simultaneous analysis of the two key components of ascorbate metabolism, ascorbic acid and dehydroascorbic acid along with ascorbic acid-2sulfate was developed, and specific aspects of sample preparation for this analysis investigated. The developed method for the analysis of the sulfide metabolome was successfully applied to the analysis of plasma from a cohort of ten healthy volunteers as a proof of concept study. This robust and versatile method could be easily applied to both existing sample cohorts and new studies, and provide new insights into sulfur redox metabolism. It could also be used in conjunction with stable isotope tracing and established methodologies for the analysis of other redox metabolomes. The analysis of polysulfides provided new insights into previously unknown sulfur related metabolic activities of the antioxidant enzyme superoxide dismutase. The method for the analysis of the ascorbate metabolome requires further work due to factors involving sample processing. Table of Contents Table of Contents ............................................................................................................... i List of Tables ................................................................................................................... vii List of Figures and Schemes.............................................................................................. ix DECLARATION OF AUTHORSHIP ..................................................................................... xxi Acknowledgements ...................................................................................................... xxiii Definitions and Abbreviations ....................................................................................... xxv Chapter 1: Introduction ........................................................................................... 1 1.1 Oxidative Stress and Redox Regulation ................................................................. 2 1.2 Redox: The H2S and NO Metabolomes.................................................................. 3 1.3 The Sulfur Redox Metabolome – Sulfide and Thiols .............................................. 4 1.3.1 Sulfide .................................................................................................... 5 1.3.2 Sulfate .................................................................................................... 6 1.3.3 Glutathione ............................................................................................ 7 1.3.4 Glutathione Cycle Thiols ......................................................................... 8 1.3.5 Cysteine .................................................................................................. 9 1.3.6 N-Acetylcysteine ................................................................................... 11 1.3.7 Homocysteine ....................................................................................... 12 1.3.8 Methanethiol ........................................................................................ 13 1.4 Analysis of Low Molecular Weight Thiols and Sulfide ......................................... 14 1.4.1 Ellman’s Reagent .................................................................................. 15 1.4.1 Sulfur Selective Electrode ..................................................................... 15 1.4.2 Methylene Blue .................................................................................... 16 1.4.3 Monobromobimane ............................................................................. 16 1.4.4 Iodoacetamide...................................................................................... 17 1.4.5 N-Ethylmaleimide ................................................................................. 18 1.5 Reactive Sulfur Species: Persulfides and Polysulfides .......................................... 19 1.5.1 Biochemistry of Polysulfides ................................................................. 19 1.5.2 Analysis of Polysulfides ......................................................................... 21 1.6 Ascorbate Metabolome ...................................................................................... 23 i 1.6.1 Ascorbic acid ........................................................................................ 23 1.6.2 Dehydroascorbic acid ........................................................................... 24 1.6.3 Ascorbic acid-2-sulfate ......................................................................... 26 1.6.4 Analysis of the Ascorbate Metabolome ................................................ 27 1.7 The NO Metabolome – Arginine and Related Metabolites ................................. 29 1.7.1 Nitric oxide ........................................................................................... 30 1.7.2 Arginine Synthesis and the Urea Cycle .................................................. 31 1.7.3 Production of NO ................................................................................. 32 1.7.4 Arginine Methylation............................................................................ 33 1.7.5 Creatine and Creatinine ........................................................................ 35 1.8 Crosstalk ............................................................................................................ 36 1.8.1 Methylation ......................................................................................... 37 1.8.2 Direct NO/H2S Interaction .................................................................... 37 1.8.1 Antioxidant Enzymes and Reactive Sulfur Species................................. 38 1.8.2 Ascorbate and Sulfur Metabolism ........................................................ 39 1.9 Liquid Chromatography and Mass Spectrometry ................................................ 40 1.10 Use of Stable Isotopes .......................................................................................
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