A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids

We have developed a novel and rapid cell-free assay of the ability of HDL t o prevent the formation of or inactivate oxidized phospholipids. HDL was te sted for its ability to inhibit the oxidation of LDL, or inhibit the oxidat ion of L-alpha -1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine ( PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PA PC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 m ug of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether t he HDL was prepared by gel electrophoresis, fast protein liquid chromatogra phy, or dextran sulfate precipitation. HDL from each of 27 patients with co ronary atherosclerosis failed to inhibit the fluorescent signal generated b y a control LDL, whereas HDL from each of 31 matched normal subjects with t he same levels of HDL cholesterol significantly inhibited the signal. Resul ts from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M . Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, H. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. J. Lipid Res. 41: 1481-1494 ) were identical. HDL from the patients also failed to inhibit the fluoresc ent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL f rom matched controls (8 of 8 patients) significantly inhibited the fluoresc ent signal.ie We conclude that this new assay has the potential to allow wi despread testing of the hypothesis that HDL that is dysfunctional in preven ting the formation or inactivating oxidized phospholipids may play an impor tant role in the development of atherosclerosis.