Evidence of modified lipoprotein in the plasma of Watanabe heritable hyperlipidemic rabbits by anion-exchange high-performance liquid chromatographic assay.

We searched for evidence for the presence of oxidatively modified low density lipoproteins (Ox-LDL) in the plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits using an anion-exchange high-performance liquid chromatographic (HPLC) method newly developed for the assay of artificially modified lipoproteins. Various Ox-LDLs were prepared by incubation of native LDL (N-LDL) from rabbit plasma with 5 microM CuCl2 for 0, 1, 3 and 24 h, and separated by anion-exchange HPLC method using a DEAE-glucomannan gel column. With an increase in the incubation time, LDL was further oxidized and retained on the DEAE-glucomannan gel. This oxidized LDL migrated faster in the anodic direction in agarose gel electrophoresis, accompanied by an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS). There was a good agreement in the chromatographic and electrophoretic behaviors of the various Ox-LDLs. LDL from WHHL rabbits showed increases in TBARS levels, electrophoretic mobility on agarose gel electrophoresis and cholesteryl ester accumulation in mouse peritoneal macrophages compared to that from normolipidemic Japanese white (JW) rabbits. When LDLs from both animals were compared by the anion-exchange HPLC method using linear gradient or stepwise elution, LDL from WHHL rabbits had a longer retention time and was eluted by a higher concentration of sodium chloride. These results suggest that LDL in WHHL rabbit plasma may undergo mild oxidative modification.