Modifications to LDL and endothelial cells by oxidation and n-3 fatty acids

Abstract

ESI-MS was used together with stable isotope labelling to determine the temporal responses of cell phosphatidylcholine (PC) composition, synthesis de novo and acyl remodelling by human umbilical vein endothelial cells (HUVEC) cultured with exogenous fatty acids. The resultant changes in cytokine product were measured. The modifications to PC composition of plasma were also measured by ESI-MS following in vivo supplementation with either DHA or EPA fatty acids for four weeks. LDL was isolated from the modified plasma and subjected to copper oxidation in the presence and absence of an Lp-PLA₂ inhibitor and the oxidised PC species produced were compared to those formed in control LDL.

These results demonstrated that HIVEC PC composition was resistant to change in response to the added fatty acids, small changes in composition were achieved primarily by acyl remodelling of existing PC species rather than by changing the specificity of synthesis de novo; changes to cytokine production in the cells was also largely unaffected. In vivo fatty acid supplementation resulted in large changes to specific PC species in plasma. However, following LDL isolation and oxidation, the same oxidised species were produced for the fatty acid modified LDLs and the control LDL. Oxidation increased three groups of PC species: lysoPC and low and high mass oxidised PC. A higher proportion of low mass oxidised species were produced in the fatty acid modified LDLs but there was no accumulation of the high mass species, which demonstrated the largest increase in the control LDL. The inclusion of the Lp-PLA₂ inhibitor in the oxidation reaction identified lysoPC and low mass oxidised PC species as the products and substrates respectively of the inhibitor. High resolution mass spectrometry allowed the exact masses and elemental formulae to be assigned for each of the oxidised species produced.

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