Generation and metabolism of 12/15-LOX esterified products
Morgan-Davies, Alwena H.
12/15-LOX is suspected to be involved in inflammatory disorders such as atherosclerosis but the exact mechanism of its action is unclear. Novel esterified products called 12- and 15- H(p)ETE-PEs were recently identified in murine macrophages and human monocytes respectively, which may at least partially account for the effects of 12/15-LOX (Maskrey et al, 2007). In this thesis the generation and metabolism of 12- and 15-H(p)ETE-PEs was characterised in murine macrophages and human monocytes. 12- and 15-H(p)ETE-PEs increased by 15 minutes following cell activation and were metabolised by three hours. However, endogenous 12- and 15-HETE-PEs increased then remained stable. Novel lipids believed to be precursors of 15-HETE-PEs, called 15-KETE-PEs, were identified in human monocytes. 15-KETE-PEs increased by 15 minutes and were metabolised by three hours and may therefore account for the temporal generation pattern of 15-H(p)ETE-PEs. 14C radiolabeled products of 15-LOX were generated and characterised in human monocytes. Detection of the 14C radiolabel in activated monocytes suggested that products of 15-LOX may form Michael adducts with proteins by three hours post activation. This may account for the 15-KETE-PE decrease in time-course samples. A method was developed to synthesise 15-H(p)ETE-PE standards, to use in a new assay for their direct quantification following MS analysis. The 15-HETE-PE standard was also used to investigate its pro- versus anti-inflammatory effect on LPS-stimulated human monocytes. 15- HETE-PE down-regulated IL-ip, TNF-a, IL-6 and G-CSF generation, suggesting that products of 15-LOX are anti-inflammatory in human monocytes. 12-H(p)ETE-PEs were externalised in activated WT macrophages, which may be important for cell-cell interactions and re-structuring the cell membrane. EM analysis indicated that 12/15-LOX"7" macrophages have a lysosomal storage disease, symptoms included the presence of lysosomal storage bodies, vacuoles and damaged mitochondria. This may be caused by detected raised levels of PE, PS, PG and PI phospholipids and cholesterol esters in 12/15-LOX"7" macrophages. The data described herein provides a platform to further investigate the function of esterified products generated by 15- and 12/15-LOX.