Feasibility of a plasma bioassay to assess oxidative protection of low-density lipoproteins by high-density lipoproteins

Abstract

Traditionally, the impact of lipoproteins on vascular disease has been evaluated in light of their quantity, that is, cholesterol content, in plasma. However, recent studies of high-density lipoproteins (HDLs) have focused on functionality with regard to atheroprotection. For example, bioassays have emerged to assess the ability of HDL, in its near native plasma environment, to promote cholesterol removal (efflux) from cells. As a result, attention has focused on developing plasma-based assays for other putative HDL protective functions including protecting low-density lipoproteins (LDLs) from oxidative damage. To determine the feasibility of such an assay in a complex sample such as plasma, we evaluated the contribution of HDL vs other plasma factors in preventing LDL oxidation. We separated normolipidemic human plasma by gel filtration chromatography and assessed each fraction for its ability to prevent LDL modification by water soluble radical and copper-initiated oxidation mechanisms. Using proteomics and selective precipitation methods, we identified major antioxidative contributions for fibrinogen, immunoglobulin G, albumin, and small soluble molecules like uric acid and ascorbate, with albumin being especially dominant in copper-initiated mechanisms. HDL particles were minor contributors (∼1%-2%) to the antioxidant capacity of plasma, irrespective of oxidation mechanism. Given the overwhelming background of antioxidant capacity inherent to highly abundant plasma proteins, specific bioassays of HDL antioxidative function will likely require its complete separation from plasma.