Bioactive lipids or lipids that activate specific signalling pathways are involved in the regulation and maintenance of normal bodily functions. Furthermore, bioactive lipid targets have been implicated in a number of conditions such as cancer, asthma and arthritis, all of which contain an inflammatory element. Additionally, a number of bioactive lipids are also known to target several different types of receptors. These include the cannabinoid receptors, peroxisome proliferator activated receptor alpha (PPARα) and transient receptor potential vanilloid-1 (TRPV1) ion channels. The assessment of bioactive lipid levels in biological systems is important for understanding their role in cell function and pathological events. The aims of this thesis were to develop an analytical method that allows the simultaneous identification and measurement of lipid mediators involved in inflammatory responses, including cyclooxygenase, lipoxygenase and cytochrome P450 metabolites, along with the endocannabinoids to determine the role of bioactive lipids in models of acute and chronic inflammatory pain. A validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the simultaneous identification and measurement of 42 bioactive lipids was developed, capable of measuring COX metabolites; PGD[subscript]2/PGE[subscript]2, TXB[subscript]2, LOX metabolites; 5-, 12-, 15-HETE, HODEs, oxoODEs, CYP metabolites; 8- and 11-HETE, EETs and the endocannabinoids; AEA and 2-AG and endocannabinoid-like compounds PEA and OEA. Levels of these analytes were quantified in rat hindpaw, dorsal root ganglia, knee joint, plasma, spinal cord and brain. This LC-MS/MS method was then used to investigate how levels of these bioactive lipids were altered in inflammation and pain. Experiments in this thesis using a PPARα competitive binding assay showed that 8-HETE and PEA bind to PPARα, along with other lipids such as fatty acids and EETs. levels of PPARα ligands were altered in the carrageenan model of inflammatory pain, suggesting that changes in bioactive lipid metabolism may influence the contribution of PPARα in inflammatory pain states. Additionally, in the same model of inflammatory pain, levels of TRPV1 ligands were altered, supporting the known role of TRPV1 in inflammatory pain states. Inhibition of 15- lipoxygenase or blockade of the metabolites resulted in attenuation of hyperalgesia, which was supported by alterations in bioactive lipid levels in vitro but not in vivo. The role of bioactive lipids in a rat model of osteoarthritis was also investigated. Levels of TRPV1 ligands were altered in this model of osteoarthritis, accompanied by the time-dependent development of pain behaviour, supporting a contribution of TRPV1 to OA-induced pain. In conclusion, the development of a novel LC-MS/MS analytical method capable of measuring a large number of bioactive lipids in vitro and in vivo, have provided novel findings to support the involvement of these lipids in inflammation and pain. Overall, these data provide evidence for the involvement of PPARα and TRPV1 ligands in inflammatory pain states.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:576503 |
| Date | January 2012 |
| Creators | Wong, Amy |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/28568/ |
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