Mass-spectrometric profiling of omega-3 polyunsaturated fatty acids and anti-inflammatory metabolites following dietary supplementation in biofluid samples and skeletal muscle cells

Inflammation is a natural biological process which acts to eliminate infectious pathogens and promote the repair of damaged tissues. The inflammatory process promotes the transport of leukocytes and plasma to the injured site or point of infection. This proactive way of an organism defending itself involves immune cells and other molecular mediators; however, when this defensive mechanism is not controlled chronic diseases including rheumatoid arthritis, coronary heart disease, cancers and many others are likely to occur. Since the study carried out in Greenland Eskimos in 1970, omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) have been known to confer positive health benefits. These essential fatty acids are made available to cells through diet and supplementation with fish oils capsules. Dietary supplementation has become necessary as these highly unsaturated fatty acids cannot be synthesised in the human body and are present at low levels in the typical Western diet. Omega-3 polyunsaturated fatty acids (PUFAs) are converted in-vivo into anti-inflammatory metabolites known as resolvins. The family of resolvins includes E and D series generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) respectively. These metabolites have been shown to be potent anti-inflammatory mediators.Projects were outlined to enhance the understanding of the incorporation of Ω-3 PUFAs into different cell types and the metabolism involved in the conversion of essential fatty acids to biologically active polyunsaturated fatty acids and further conversion through the different metabolism pathways to their corresponding lipid mediators. All projects were carried out as part of the Translational Chemical Biology group at Loughborough University, with the collective focus on the chemical synthesis and biological evaluation of novel naturally informed anti-inflammatory agents.A gas chromatography mass spectrometry method was developed and validated to determine the incorporation of biologically active fatty acids after oral supplementation. Incorporation of these polyunsaturated fatty acids in different cell types was considered. Samples considered include peripheral blood mononuclear cells (PBMCs), red blood cells and dried blood spots. Literature on incorporation of omega-3 fish oils reports maximal incorporation in peripheral blood mononuclear cells after months of oral supplementation, this was confirmed. However, this report was further investigated incorporation into PBMCs and data indicates incorporation occurs within the first seven days of supplementation.In red blood cells, incorporation occurs over a longer period and it takes up to 8 weeks for the maximum levels to be reached. This suggests that the lifetime of the cells in-vivo plays a role in how long it takes for full incorporation to be achieved. Dried blood spots were shown to incorporate in a similar manner to red blood cell samples, which suggests they could act as a surrogate and is more easily transportable analogue to venous blood sampling that gives a more accurate view of the tissue PUFA status than PBMC and plasma samples. Data from this study also suggests that PBMC stores an amount of Ω -6 AA as there was no change even though participants were placed on a restricted diet as part of the experimental design. Following the investigation into incorporation of Ω-3 fish oils in different cell types after oral supplementation, a liquid chromatography-mass spectrometry method was developed to identify the anti-inflammatory metabolites 18-HEPE and resolvin E1 after exposing human skeletal muscle cells to inflammatory and non-inflammatory conditions in the presence of EPA with/without aspirin. This was to ascertain the activation of the different metabolic pathways of converting biologically active EPA to its anti-inflammatory lipid metabolite resolvin E1. Identification of 18-HEPE and possible Resolvin E1 in both conditions indicated the activation of the cytochrome PY450 and aspirin triggered COX-2 metabolic pathways. The key finding of this research was that 18-HEPE was produced in large quantities in the extracellular medium with and without the presence of aspirin. These data suggest that cytochrome P450 plays a key role in the enzymatic pathway leading to the resolvin family and that 18-HEPE can be either exported from the cells or formed by enzyme action at the cell wall.