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 hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE-glucomannan gel, and direct CL detection of HPOs 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, were essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE-glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.
Yamaguchi
Y
Y
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Hyogo, Japan. yusan@mwu.mukogawa-u.ac.jp
Kagota
S
S
Kunitomo
M
M
Haginaka
J
J
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006861:Hydrogen Peroxide / Q000032:analysis*; D006949:Hyperlipidemias / Q000097:blood / Q000235:genetics; D008074:Lipoproteins / Q000737:chemistry*; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D011817:Rabbits; D015203:Reproducibility of Results
D008074:Lipoproteins; D006861:Hydrogen Peroxide