Meyer, David Ford; (1994) Analysis of the structural changes that occur during the oxidation of human low density lipoproteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Elevated levels of low density lipoproteins (LDL) in plasma correlate with increased atherosclerosis and risk of cardiovascular disease. Oxidation of LDL is thought to be an important event in this process. Analytical techniques in conjunction with X-ray and neutron solution scattering were used to study the time course of biochemical and structural changes which occur during the oxidative modification of LDL in the presence of copper ions. Neutron scattering provided information on the overall structure and size of the lipoproteins. Data from many LDL samples showed that native LDL had a polydisperse spherical structure. X-ray scattering data provided information on the internal structure of LDL. At temperatures below the transition temperature of the lipids, this consisted of two concentric shells of cholesterol esters and triglycerides, surrounded by a monolayer of phospholipid and protein. Conditions were also optimised for monitoring oxidation experiments by solution scattering techniques. Chemical analysis indicated that arachidonic acid is preferentially oxidised before the formation of maximal lipid hydroperoxides. High performance thin layer chromatography showed that phosphatidylcholine is continuously converted to lysophosphatidylcholine throughout the oxidation. This indicated the presence of an intrinsic phospholipase-A2 like activity in LDL. Core lipids were not oxidatively modified until 12 - 15 h after initiation of oxidation. The uptake of oxidised LDL by macrophages and the formation of apoB aggregates (seen by SDS-PAGE) are affected in a Cu2+ dependent manner. Neutron scattering indicated that during oxidation of LDL, aggregation had occurred, as evidenced by increases in the Guinier parameters. These agree with the changes observed by SDS electrophoresis studies. Analysis of polydispersity indicated that only a small proportion of LDL underwent aggregation. X-ray scattering indicated that the internal region attributed to the phospholipid hydrocarbon chains increased in electron density throughout the time course of oxidation. This was interpreted to represent the incorporation of oxygen atoms into peroxidised polyunsaturated fatty acids. Furthermore as the oxidation progressed the core lipids became increasingly disordered. The X-ray scattering is consistent with the time course of arachidonic acid oxidation and HPTLC analysis. The structural changes matched the compositional analysis which suggested that oxidation may progress from the outside towards the internal core of LDL.

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