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Detection of Stress- and Taste-Related Compounds in Human Saliva

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Detection of Stress- and Taste-Related Compounds in Human Saliva Detection of stress- and taste-related compounds in human saliva Truc Van Nguyen Biotechnology Submission date: October 2018 Supervisor: Oleksandr Dykyy, IBT Co-supervisor: Elena Shumilina, IBT Norwegian University of Science and Technology Department of Biotechnology and Food Science Acknowledgement This thesis was conducted and written at the Department of Biotechnology and Food Science under Norwegian University of Technology and Science (NTNU), Trondheim – Norway. This thesis is a closure for my 2-years master program in Biotechnology, at NTNU. Firstly, I would like to thank my co-supervisor Elena Shumilina for her extremely kindness and patience. Without her helpful guiding and advising, I would never finish this work. Next, I would like to acknowledge Professor Alexander Dikiy for presenting me chance to be a part of the research group. I also would like to forward my appreciation to my lab-friend Ida Møller, my junior Huong Nguyen, and all my fellow friends for their cooperation, encouragement throughout this project. Especially, I would like to give my gratefulness to my husband Mr. Truls Rasmussen for motivating, supporting, and taking care of me every single day. Finally, my special thank is given to my beloved family with my deepest gratitude. Without their love and encouragement, I would have never been able to study in NTNU nor finish this thesis. Truc Van Nguyen Trondheim, September 2018 I II Abstract Saliva is made up by 99% water and only 1% other components including plasma-derived ions, proteins, hormones and so on. Hence, even though they are included in only 1% composition of saliva, those components have a vital role as biomarkers for such a huge range of molecules. The hypothesis for this thesis is that it is possible to detect cortisol-like compound (CLC) and taste-related compound (TRC) in saliva using NMR approach. With CLCs, cortisol, cortisone, aldosterone, and corticosterone are performed with several experiments. With TRCs, adenosine-related compounds and other taste compounds are analysed. Through a series of experiments, 5.8 ppm region in 1D spectra is suggested as the most promising region to detect CLC in human and animal saliva. NMR approaching can also distinguish sample’s genders, as well as detect if a person uses corticosteroid. Salmon extraction using saliva together with TCA are used in an attempt to extract flavouring compounds. On one hand, TCA extraction result shows greater stability than extraction result performing with saliva. On the other hand, saliva quality and condition have such an influence in the extraction result. In short, the possibility to track the concentration of many TRCs in human saliva over time is confirmed. III IV Abbreviations Acronym Description µL Microlitre ACTH Adrenocorticotropic Hormone ADP Adenosine 5’-iphosphate AMP Adenosine 5’-monophosphate ANS Autonomic Nervous System ATP Adenosine 5’-triphosphate BMRB Biological Magnetic Resonance Data Bank CLC Cortisol-like compound CNS Central Nervous System COPD Chronic Obstructive Pulmonary Disease CRF Corticotropin-Releasing Factor D2O Deuterated water DEPT Distortionless Enhancement by Polarization Transfer Epp. tube Eppendorf tube g Gram G G-force or Relative Centrifugal Force (RCF) GAS General Adaptation Syndrome GLC Gas-Liquid Chromatography V HMBC Heteronuclear Multiple Bond Correlation HMDB The Human Metabolome Database HPA Hypothalamic–Pituitary–Adrenal axis hrs Hours HSQC Heteronuclear Single Quantum Correlation Hx Hypoxanthine IMP Inosine monophosphate Ino Inosine mM Millimolar or millimole per litre mmol Millimole NMR Nuclear Magnetic Resonance NTNU Norwegian University of Technology and Science PaNS Parasympathetic Nervous System PCA Principal Component Analysis PNS Peripheral Nervous System ppm Part Per Million Sal Saliva SalmA Salmon brand that was used for this project SNS Somatic Nervous System SRF Somatotropin-Releasing Factor VI SyNS Sympathetic Nervous System TCA Trichloroacetic acid TOCSY TOtal Correlation SpectroscopY TSP 3-(Trimethylsilyl)propanoic acid Xan Xanthine VII VIII Table of Contents Acknowledgement ..................................................................... I Abstract ................................................................................. III Abbreviations ............................................................................ V Table of Contents .................................................................... IX Introduction ......................................................................... 1 1.1 Stress ............................................................................... 2 1.1.1 History and definition .................................................... 2 1.1.2 Biology of stress ........................................................... 4 1.1.3 Cortisol and cortisol-like compounds (CLC) ....................... 9 1.2 Biofluid metabolomic ........................................................ 11 1.3 Taste .............................................................................. 12 1.4 Salmon source ................................................................. 14 1.5 Nuclear Magnetic Resonance (NMR) .................................... 15 1.5.1 One-Dimensional (1D) NMR spectroscopy ...................... 16 1.5.2 Two-Dimensional (2D) NMR spectroscopy ...................... 16 1.6 Objectives of the project ................................................... 18 Methodology ...................................................................... 19 2.1 Experimental design ......................................................... 19 2.2 Chemicals and Equipment ................................................. 19 IX 2.3 CLC stock solution ............................................................. 21 2.4 CLC titration preparation .................................................... 22 2.5 Standard saliva sampling ................................................... 23 2.6 Salmon extraction in saliva (SalmA-Sal) .............................. 23 2.7 Salmon extraction in TCA (SalmA-TCA)................................ 24 2.8 Other experiments ............................................................ 24 2.8.1 Salmon-TCA-saliva (SalmA-TCA-Sal) interaction .............. 24 2.8.2 Titration of metabolite .................................................. 24 2.8.3 Student saliva ............................................................. 25 2.9 Sample preparation for NMR analysis .................................. 25 2.10 NMR experiments .............................................................. 25 2.11 NMR processing ................................................................ 26 2.12 Analysis ........................................................................... 26 2.12.1 CLC assignment .......................................................... 26 2.12.2 Statistical analysis ....................................................... 27 Result ................................................................................ 28 3.1 Error calculating ............................................................... 28 3.1.1 Pipetting error ............................................................. 28 3.1.2 TSP concentration uncertainty ....................................... 29 3.1.3 NMR – S/N measurement ............................................. 29 X 3.1.4 Integration uncertainty ................................................ 29 3.1.5 Metabolite quantification .............................................. 30 3.2 Saliva composition............................................................ 31 3.3 CLC assignment with stock solutions ................................... 32 3.3.1 Hydrocortisone ........................................................... 32 3.3.2 Cortisone ................................................................... 34 3.3.3 Aldosterone ................................................................ 37 3.3.4 Corticosterone ............................................................ 40 3.4 Human saliva titration with CLC standard ............................ 44 3.4.1 Hydrocortisone titration ............................................... 44 3.4.2 Cortisone titration ....................................................... 46 3.4.3 Aldosterone titration .................................................... 48 3.4.4 Corticosterone titration ................................................ 50 3.5 Study of student saliva ..................................................... 54 3.6 Taste-related compounds in SalmA extract .......................... 58 3.6.1 Adenosine-related compounds ...................................... 58 3.6.2 Other taste-related compounds .................................... 67 Conclusion .......................................................................... 74 References ......................................................................... 77 Appendices ......................................................................... 83 XI XII Introduction Stress has become one of the most common terms that has been mentioning in everyday life. It is a serious problem, especially in developed countries where numerous stress-causing factors can be found in almost every aspect of such a busy life. When someone says “I am stressed”, it simply exposes how she or he feels. More than just an emotional expression, stress is a disorder indicated by the significant change of some metabolite molecules in our body in order to response to the change of various factors. The result of
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