Nuevos abordajes de cuantificación en lipidómica y su aplicación en la esclerosis múltiple

Abstract

The study of lipid metabolism has a long tradition in biomedicine, it is enough to remember the great variety of pathophysiological functions and implications granted to lipid classes such as cholesterol and triglycerides. However, for decades, the study of lipids has been limited to its study as class or group- in the case of triglycerides, a skeleton of glycerol replaced by three fatty acid chains-, obviating that each class is formed by a huge diversity of species derived from the combination of different fatty acids, with a variable length and unsaturation degree. As species, lipids are the most abundantmolecules in quantity and variety of the metabolome. The traditional experimental approach to study the fatty acid composition of a lipid class involves its separation by some chromatographic technique, its hydrolysis and the subsequent analysis of the fatty acid composition. These procedures are long and with limited reproducibility. Fortunately, the advances in mass spectrometry, in parallel to the availability of bioinformatics tools that allow the analysis of large amounts of data, have facilitated the study of the different lipid species present in a sample, giving rise to a new discipline, lipidomics. Lipidomics is essentially a multidisciplinary field, it uses experimental techniques of some complexity such as mass spectrometry, without forgetting that lipidomics responds to the attempt to fully characterize the lipid set of an organism or biological system and its relationship with other components and cellular events. In the first work of this thesis we have studied the possibilities that MALDI-TOF technology (“Matrix-Assisted Laser Desorption/Ionization with Time-of-flight detector”) offers in the field of lipidomics. MALDI is a mass spectrometry technique widely used in proteomics and glycolics for characterization and identification. However, the quantitative possibilities of MALDI, which are essential in lipidomics, have been poorly studied. In this study, we have optimized and validated a working methodology that allows the characterization and quantification of more than 150 lipid species in plasma, belonging to 7 classes: triglycerides, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin and sulfatides. The great variety and abundance of lipids at the brain means that changes in their concentration or defects in metabolic pathways have a significant impact on brain function. In fact, the alterations in lipid metabolism are present in several neurological diseases, such as Parkinson's disease, Alzheimer's disease or Multiple Sclerosis (MS). MS is a neurodegenerative disease caused by primary destruction of myelin, affecting nerve conduction. The application of lipidomics represents an opportunity to study the role of different lipid species in MS.In the second work of this thesis, we have conducted a lipidomic study to characterize the effect of ellagic acid treatment in rats with experimental autoimmune encephalomyelitis. Experimental autoimmune encephalomyelitis (EAE) is the main animal experimental model used for the study of MS. Ellagic acid is a polyphenol that is known for its antioxidant effect and suppressive effect of neuroinflammation. In the study we show that treatment with ellagic acid reduces the loss of sphingolipids in cerebral cortex and spinal cord associated with acute outbreaks of EAE. The changes in the concentration of sphingolipids (hexosylceramides, sulfatides, sphingomyelin, etc.), all of them present in myelin sheaths, correlate with the levels of myelin basic protein, a marker of myelin integrity. In view of these results, we demonstrate in C6 (rat glioma) and HOG (human oligodendroglioma) cell models that some metabolites derived from ellagic acid increase sphingolipid synthesis. MS is an entity with variable clinical presentation and its diagnosis is difficult. There are no precise biomarkers of diagnosis and evolution. In the third work, of this thesis we have studied if the quantification of sphingolipids in cerebrospinal fluids is of utility in a cohort of MS patients. According to our results, the quantification of sphingolipids in cerebrospinal fluid in patients in remission phase is not very useful in this pathology.