Background-Electronegative LDL [LDL(Ϫ)], a modified subfraction of LDL present in plasma, induces the release of interleukin-8 and monocyte chemotactic protein-1 from cultured endothelial cells. Methods and Results-We demonstrate that platelet-activating factor acetylhydrolase (PAF-AH) is mainly associated with LDL(Ϫ). LDL(Ϫ) had 5-fold higher PAF-AH activity than the nonelectronegative LDL subfraction [LDL(ϩ)] in both normolipemic and familial hypercholesterolemic subjects. Western blot analysis after SDS-PAGE confirmed these results, because a single band of 44 kDa corresponding to PAF-AH appeared in LDL(Ϫ) but not in LDL(ϩ).Nondenaturing polyacrylamide gradient gel electrophoresis demonstrated that PAF-AH was bound to LDL(Ϫ) regardless of LDL size. In accordance with the above findings, nonesterified fatty acids, a cleavage product of PAF-AH, were increased in LDL(Ϫ) compared with LDL(ϩ). Conclusions-The

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.

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