Please use this identifier to cite or link to this item: http://hdl.handle.net/1946/8146
Cancer cells show many differences from healthy normal cells in their metabolism, which contribute to their survival and growth. Lipids have numerous of different functions in biological processes, structural as well as regulatory. Fatty acid synthase (FAS) is an essential enzyme with the final product palmitic acid (hexadecaenoic acid). High levels of FAS is a characteristic of many human malignancies. Hydroxyeicosatetraenoic acids (HETEs) are the products of lipoxygenase (LOX) pathway and have been implicated in cancer development. The lichen secondary metabolite protolichesterinic acid (PA) is a potent inhibitor of 5- and 12-LOX and may also have FAS inhibitory activity. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique that can be used in targeted lipid analysis and quantification of lipid metabolites.
The aims of this project were to develop a sample preparation technique and validated LC-MS/MS method for the quantification of palmitic acid and HETEs in two human cancer cell lines, SK-Br3 from breast cancer and Capan-2 from pancreatic cancer. Protolichesterinic acid was purified with preparative high-pressure liquid chromatography (HPLC). Solid-phase extraction method was employed to extract HETEs.
Palmitic acid could not be successfully analysed because of contamination problems. An intraday validation assessment showed that the quantitative determination is linear for LTB4, 12- and 5-HETE in the range tested (120-40000 pg/mL), and a coefficient of variation lower than 15% was obtained for samples analysed. Extraction efficiency from standard solutions was poor for LTB4, 12- and 5-HETE (63.9%, 38.5%, and 33.5% respectively).
Extraordinary precautions should be taken to achieve accurate analysis of palmitic acid and to avoid fatty acid contamination. A LC-MS/MS method was validated for the quantification of LOX pathway products. Multiple samples and further development of sample preparation is needed for quantification of LTB4, 12- and 5-HETE in Capan-2 cancer cells. Quantification of lipids from cultured cancer cells is necessary for future research on this important aspect of cancer cell metabolism and drug effects.
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