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Curriculum: Biology and Marine Ecology) UNIVERSITÀ POLITECNICA DELLE MARCHE Department of Life and Environmental Sciences PhD course Life and Environmental Science (curriculum: Biology and Marine Ecology) Fatty acids as biomarkers of the influence of biological and environmental factors on marine organisms PhD student: Tutor: Matteo Antonucci Cristina Truzzi Academic Year 2017/2018 Index 1 Introduction .............................................................................................................. 1 2 Lipids ........................................................................................................................ 3 2.1 Lipids classification ............................................................................................ 3 2.1.1 Acylglycerols ........................................................................................... 3 2.1.2 Phospholipids .......................................................................................... 4 2.1.3 Sphingolipids ........................................................................................... 5 2.1.4 Glycolipids ............................................................................................... 5 2.1.5 Steroids ................................................................................................... 5 2.1.6 Waxes ..................................................................................................... 6 2.2 Fatty acids .......................................................................................................... 6 2.2.1 Nomenclature .......................................................................................... 7 2.2.2 Classification ........................................................................................... 7 2.2.3 Properties ................................................................................................ 8 2.2.4 Metabolism .............................................................................................. 9 2.2.4.1 De novo synthesis of fatty acids ............................................... 9 2.2.4.2 Catabolism ................................................................................ 9 2.3 Membrane, fatty acids and temperature .......................................................... 10 2.3.1 Lipid bilayer ........................................................................................... 11 2.3.2 Effects of temperature on biological membranes .................................. 11 2.3.3 Thermal perturbation of membrane structure and function .................... 11 2.3.4 Membrane remodeling: the basis of thermal adaptation ........................ 12 3 Fishes ..................................................................................................................... 14 3.1 Distribution and morphology of teleostei .......................................................... 14 3.2 Muscle ............................................................................................................. 14 3.3 Liver ................................................................................................................. 15 3.4 Gills .................................................................................................................. 17 3.5 Lipids in fish ..................................................................................................... 18 3.6 Metabolism of fatty acids in fish ....................................................................... 19 3.7 Lipid digestion and absorption in fish ............................................................... 20 3.8 Role of fatty acids in fish .................................................................................. 22 3.9 Antarctic fishes ................................................................................................. 23 3.9.1 Warm acclimation response in Antarctic fishes ..................................... 24 i 4 Fatty acids as biomarkers ...................................................................................... 26 5 Quantification of fatty acids in the muscle of Antarctic fish Trematomus bernacchii by gas chromatography-mass spectrometry: Optimization of the analytical methodology ........................................................................................................... 29 5.1 Abstract ............................................................................................................ 29 5.2 Introduction ...................................................................................................... 29 5.3 Material and methods ...................................................................................... 32 5.3.1 Sample collection .................................................................................. 32 5.3.2 Extraction of lipids ................................................................................. 32 5.3.3 Preparation of fatty acid methyl esters (FAMEs) .................................... 33 5.3.4 Instrumentation and analytical conditions .............................................. 33 5.3.5 Method performance ............................................................................. 34 5.3.6 Statistical analysis ................................................................................. 35 5.4 Results and discussion .................................................................................... 35 5.4.1 Lyophilization step ................................................................................. 35 5.4.2 Optimization of GC-MS method ............................................................. 35 5.4.3 Performance data .................................................................................. 36 5.4.4 Fatty acid quantification ......................................................................... 37 5.5 Conclusions ..................................................................................................... 39 5.6 Acknowledgements .......................................................................................... 40 5.7 Figures, Tables and Supplementary materials ................................................. 41 5.8 References ........................................................................................................ 53 6 Heat shock influences the fatty acid composition of the muscle of the Antarctic fish Trematomus bernacchii ............................................................................................................. 59 6.1 Abstract ............................................................................................................ 59 6.2 Introduction ...................................................................................................... 59 6.3 Material and methods ...................................................................................... 61 6.3.1 Chemicals .............................................................................................. 62 6.3.2 Sample collection and experimental design .......................................... 62 6.3.3 Lipid extraction and fatty acid analysis .................................................. 62 6.3.4 Statistical analysis ................................................................................. 63 6.4 Results ............................................................................................................. 64 6.4.1 Lipid content .......................................................................................... 64 6.4.2 FAs composition .................................................................................... 64 ii 6.5 Discussion ........................................................................................................ 66 6.6 Conclusions ..................................................................................................... 68 6.7 Acknowledgements .......................................................................................... 69 6.8 Figures, Tables and Supplementary materials ................................................. 70 6.9 References ........................................................................................................ 76 7 Gas chromatography–mass spectrometry analysis on effects of thermal shock on the fatty acid composition of the gills of the Antarctic teleost, Trematomus bernacchii ............................................................................................................... 84 7.1 Abstract ............................................................................................................ 84 7.2 Introduction ...................................................................................................... 85 7.3 Material and methods ...................................................................................... 88 7.3.1 Chemicals .............................................................................................. 88 7.3.2 Sample collection and experimental design .......................................... 88 7.3.3 Ethics ..................................................................................................... 89 7.3.4 Lipid extraction and fatty acid analysis .................................................. 89 7.3.5 Statistical analysis ................................................................................. 90 7.4 Results and discussion ...................................................................................
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