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 μM 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.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Goldstein J.L.
        • Ho Y.K.
        • Basu S.K.
        • et al.
        Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.
        Proc Natl Acad Sci USA. 1979; 76: 333-337
        • Steinberg D.
        • Parthasarathy S.
        • Carew T.E.
        • et al.
        Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenecity.
        New Engl J Med. 1989; 320: 915-924
        • Yokode M.
        • Kita T.
        • Arai H.
        • et al.
        Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract.
        Proc Natl Acad Sci USA. 1987; 85: 2344-2348
        • Boyd H.C.
        • Gown A.M.
        • Wolfbauer G.
        • et al.
        Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit.
        Am J Pathol. 1989; 135: 815-825
        • Haberland M.E.
        • Fong D.
        • Cheng L.
        Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits.
        Science. 1988; 241: 215-218
        • Kotani K.
        • Maekawa M.
        • Kanno T.
        • et al.
        Distribution of immunoreactive malondialdehyde-modified low-density lipoprotein in human serum.
        Biochim Biophys Acta. 1994; 1215: 121-125
        • Kanazawa T.
        • Uemura T.
        • Osanai T.
        • et al.
        Plasma peroxidized low-density lipoprotein with hydroperoxidized cholesteryl linoleates estimated in patients with familial hypercholesterolemia.
        Pathobiology. 1994; 62: 269-282
        • Miyazawa T.
        • Fujimoto T.K.
        • Koikawa S.
        Determination of lipid hydroperoxides in low density lipoprotein from human plasma using high performance liquid chromatography with chemiluminescence detection.
        Biomed Chromatogr. 1990; 4: 131-134
        • Hodis H.N.
        • Kramsch D.M.
        • Pietro A.
        • et al.
        Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL−).
        J Lipid Res. 1994; 35: 669-677
        • Haginaka J.
        • Yamaguchi Y.
        • Kunitomo M.
        Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.
        Anal Biochem. 1995; 232: 163-171
      1. Yamaguchi Y, Kunitomo M, Haginaka J. Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins of rabbits, rats and mice. J Chromatogr B (submitted).

        • Hatch F.T.
        • Lees R.S.
        Practical methods for plasma lipoprotein analysis.
        Adv Kuoud Res. 1968; 6: 1-68
        • Morita H.
        • Kitazawa A.
        • Tomoda T.
        Absorption and separation of biopolymer using DEAE-glucomannan gel.
        Chromatography. 1990; 11: 20-21
        • Yokode M.
        • Kita T.
        • Arai H.
        • et al.
        Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract.
        Proc Natl Acad Sci USA. 1987; 85: 2344-2348
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • et al.
        Protein measurement with the folic phenol reagent.
        J Biol Chem. 1951; 193: 265-275
        • Kunitomo M.
        • Yamaguchi Y.
        • Matsushima K.
        • et al.
        Cholesterol metabolism in serum and aorta of inbred mice fed a high-cholesterol diet.
        Japan J Pharmacol. 1984; 34: 153-158
        • Yagi K.
        A simple fluorometric assay for lipoperoxide in blood plasma.
        Biochem Med. 1976; 15: 212-216
        • Noble R.P.
        Electrophoretic separation of plasma lipoproteins in agarose gel.
        J Lipid Res. 1986; 9: 693-700
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature (London). 1970; 227: 680-685
        • Narayan K.A.
        • Narayan S.
        • Kummerow F.A.
        Disk electrophoresis of human serum lipoproteins.
        Nature. 1965; 205: 246-248
        • Cazzolate G.
        • Avogaro P.
        • Bittolo-Bon G.
        Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC.
        Free Radic Biol Med. 1991; 11: 247-253
        • Demuth K.
        • Myaram I.
        • Chappey B.
        • Vedie B.
        • Pech-Amesellem M.A.
        • Haberland M.E.
        • Moatti N.
        A cytotoxic electronegative LDL subfraction is present in human plasma.
        Arterioscler Thromb Vasc Biol. 1996; 16: 773-783
        • Bittolo-Bon S.A.
        • Cazzolate G.
        • Hodis H.
        • Hwang J.
        • Zamburlini A.
        • Maiorino M.
        • Ursini F.
        LDL− is a lipid hydroperoxide-enriched circulating lipoprotein.
        J Lipid Res. 1997; 38: 419-428
        • Buja L.M.
        • Kita T.
        • Goldstein J.L.
        • et al.
        Cellular pathology of progressive atherosclerosis, in the WHHL rabbit: an animal model of familial hypercholesterolemia.
        Arteriosclerosis. 1983; 3: 87-101
        • Williams R.J.
        • Motteram J.M.
        • Sharp C.H.
        • et al.
        Dietary vitamin E and the attenuation of early lesion development in modified Watanabe rabbits.
        Atherosclerosis. 1992; 94: 153-159
        • Nagano Y.
        • Nakamura T.
        • Matsuzawa Y.
        • et al.
        Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit: long-term antiatherogenic effect and effects on established plaques.
        Atherosclerosis. 1992; 92: 131-140
        • Kita T.
        • Yokode M.
        • Watanabe Y.
        • et al.
        Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia.
        J Clin Invest. 1986; 77: 1460-1465