High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlip idemic (WHHL) rabbits. The method involves anion-exchange HPLC separation i n N- and Ox-LDLs using a DEAE-glucomannan gel, and direct CL detection of H POs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100 , which could solubilize lipids, was essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was mo re retained on the DEAE-glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HFO levels in N- and Ox -LDLs of WHHL rabbits without extraction of the lipids. (C) 1999 Elsevier S cience B.V. All rights reserved.