TIME-COURSE STUDIES BY NEUTRON SOLUTION SCATTERING AND BIOCHEMICAL ASSAYS OF THE AGGREGATION OF HUMAN LOW-DENSITY-LIPOPROTEIN DURING CU2-INDUCED OXIDATION()

The oxidative modification of low-density lipoproteins (LDL) is recogn ized to be a key event in the development of atherosclerotic plaques o n artery walls. The characteristics of LDL oxidized by cells of the ar tery wall can be imitated by the addition of Cu2+ ions to initiate lip id peroxidation in LDL. Neutron scattering of LDL in (H2O)-H-2 buffers enables the time course of changes in the gross structure of LDL duri ng oxidation to be continuously monitored under conditions close to ph ysiological. Oxidation of LDL [2 mg of apolipoprotein B (apoB) protein /ml] was studied in the presence of 6.4, 25.6 and 51.2 mu mol of Cu2+/ g of apoB by incubation at 37 degrees C for up to 70 h. Neutron Guinie r analyses showed that the radius of gyration R(G) (indicative of size ) and the forward-scattered intensity at zero angle I(0) (indicative o f M(r)) continuously increased during oxidation, indicating that LDL h ad aggregated. Both the rate of aggregation and the change in R(G) and I(0) values after 10 and 50 h increased with Cu2+ concentration. Dist ance-distribution functions P(r) showed that, within 4 h, the maximum dimension of LDL increased from 23 to 55 nm. The P(r) curves of oxidat ively modified LDL exhibited two peaks at 10-12 nm and 26 nm. The 10-1 2 nm peak corresponds to native LDL, and the 26 nm peak is assigned to the initial formation of LDL dimers and trimers and their progression to form higher oligomers. The growth of the 26 nm peak depended on Cu 2+ concentration. Particle-size-distribution functions D-v(r) suggeste d that the polydisperse spherical structure of LDL ceased to exist aft er 30 h, at which point the LDL samples underwent a phase separation. Related, but not identical, changes in the I(Q) and P(r) curves were o bserved when native LDL was self-aggregated by brief vortexing. Parall el assessment of LDL protein modification by SDS/PAGE showed increased aggregation and degradation of apoB with increased Cu2+ concentration s, and that the main apoB protein band had diminished after 2-8 h, dep ending on the amount of Cu2+ added. The uptake and degradation of oxid ized I-125-labelled LDL by mouse peritoneal macrophages occurred maxim ally within the first 10 h, and increased in proportion to the Cu2+ co ncentration. ApoB protein broke down within the first 10 h of oxidatio n, and this is the period when scavenger receptors on macrophages can recognize and internalize oxidized LDL. Within 10 h, the protein-lipid interactions responsible for the spherical LDL structure became desta bilized by protein fragmentation. After 30 h, LDL formed large vesicul ar aggregates with no regular structure. In summary, neutron-scatterin g data show that LDL aggregation is continuous during oxidation.