Advertisement

Isolation, purification, and characterization of a lipoprotein containing apo B from the human aorta

  • Henry F. Hoff
    Correspondence
    Reprint requests should be addressed to: Henry F. Hoff, Ph.D., Department of Atherosclerosis, Division of Research, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44106, U.S.A.
    Affiliations
    Department of Medicine, Baylor College of Medicine, Houston, TX 77030 U.S.A.
    Search for articles by this author
  • John W. Gaubatz
    Affiliations
    Department of Medicine, Baylor College of Medicine, Houston, TX 77030 U.S.A.
    Search for articles by this author
      This paper is only available as a PDF. To read, Please Download here.

      Abstract

      LDL-like lipoproteins were extracted from a buffer-soluble fraction of homogenates of both grossly normal intima and fatty-fibrous plaques from the human aorta. A low-speed supernate of such homogenates was subjected to differential ultracentrifugation to isolate a d 1.006–1.063 density fraction which was further purified by gel filtration on agarose columns, or by affinity chromatography of low-speed supernatants of aortic homogenates on immunoabsorbents (anti-apo B). All recovered immunoreactivity for apo B as measured by electroimmunoassay was found in the retarded fraction following gel filtration or affinity chromatography. Characterization of the aorta-extracted lipoprotein containing apo B illustrated that the lipoprotein was quite similar to plasma LDL with respect to lipid composition, lipid/protein ratio, identification of apo B by polyacrylamide gel electrophoresis, and particle size by electron microscopy. However, aorta-derived lipoproteins differed from plasma LDL with respect to fatty acid composition and electrophoretic mobility. They showed an increase in stearate content which was most prominent in the cholesteryl ester and triglyceride fractions, as well as an increase in oleate and a reduction in linoleate content in both cholesteryl ester and phospholipid fractions. During purification aorta-derived lipoproteins maintained an increase in electrophoretic mobility compared to plasma LDL. This increase in surface charge might be a result of the demonstrated contamination with sulfated glycosaminoglycans (1.5% by weight). However, digestion with chondroitinase ABC had no effect on electrophoretic mobility. The lipoproteins extracted and purified from grossly normal intima and fatty-fibrous plaques possessed the same similarities as well as differences in characteristics relative to plasma LDL. The results from this study suggest that the lipoproteins extracted from the human aorta may represent either the preferential retention of a subclass of plasma LDL with slightly different characteristics from the average but with greater affinity for intimal material, or the products of modification of normal plasma LDL following retention by extracellular components of the aortic intima. This modification may be due to complex formation of LDL with such components.

      Keywords

      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:

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

      References

        • Hoff H.F.
        • Jackson R.L.
        • Mao S.J.T.
        • Gotto Jr., A.M.
        Localization of low-density lipoproteins in atherosclerotic lesions from human normolipemics employing a purified fluorescent-labeled antibody.
        Biochim. Biophys. Acta. 1974; 351: 407
        • Hoff H.F.
        • Lie J.T.
        • Titus J.L.
        • Bayardo R.J.
        • Jackson R.L.
        • DeBakey M.E.
        • Gotto Jr., A.M.
        Lipoproteins in atherosclerotic lesions-Localization by immunofluorescence of apo-low density lipoproteins in human atherosclerotic arteries from normal and hyperlipoproteinemics.
        Arch. Path. 1975; 99: 253
        • Hoff J.F.
        • Lie J.T.
        • Titus J.L.
        • Jackson R.L.
        • DeBakey M.E.
        • Bayardo R.J.
        • Kim H.S.
        • Gotto Jr., A.M.
        The localization patterns of plasma apolipoproteins in human atherosclerotic lesions.
        Circ. Res. 1975; 37: 72
        • Walton K.W.
        • Williamson N.
        Histological and immunofluorescent studies on the human atheromatous plaque.
        Atherosclerosis. 1968; 8: 599
        • Kao V.C.
        • Wissler R.W.
        Study of the immunohistochemical localization of serum lipoproteins and other plasma proteins in human atherosclerotic lesions.
        Exp. Mol. Path. 1965; : 465
        • Hoff H.F.
        • Heideman C.L.
        • Gaubatz J.W.
        • Gotto Jr., A.M.
        • Erickson E.E.
        • Jackson R.L.
        Quantification of apolipoprotein B in grossly normal human aorta.
        Circ. Res. 1977; 40: 56
        • Hoff H.F.
        • Heideman C.L.
        • Gotto Jr., A.M.
        • Gaubatz J.W.
        Apolipoprotein B retention in the grossly normal and atherosclerotic human aorta.
        Circ. Res. 1977; 41: 684
        • Hoff H.F.
        • Heideman C.L.
        • Gaubatz J.W.
        • Scott D.W.
        • Gotto Jr., A.M.
        Detergent extraction of tightly-bound apo B from extracts of normal intima and plaques.
        Exp. Mol. Path. 1978; 38: 290
        • Hoff H.F.
        • Heideman C.L.
        • Gaubatz J.W.
        • Scott D.W.
        • Titus J.L.
        • Gotto Jr., A.M.
        Correlation of apolipoprotein B retention with the structure of atherosclerotic plaques from human aortas.
        Lab. Invest. 1978; 38: 560
        • Hoff H.F.
        • Heideman C.L.
        • Gaubatz J.W.
        • Titus J.L.
        • Gotto Jr., A.M.
        Quantitation of apo B in human aortic fatty streaks-A comparison with grossly normal intima and fibrous plaques.
        Atherosclerosis. 1978; 30: 263
        • Hoff H.F.
        • Karagas M.
        • Heideman C.L.
        • Gaubatz J.W.
        • Gotto Jr., A.M.
        Correlation in the human aorta of apo B fractions with tissue cholesterol and collagen content.
        Atherosclerosis. 1979; 32: 259
        • Smith E.B.
        • Slater R.S.
        The chemical and immunological assay of low-density lipoproteins extracted from human aortic intima.
        Atherosclerosis. 1970; 11: 417
        • Smith E.B.
        • Massie I.B.
        • Alexander K.M.
        The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin.
        Atherosclerosis. 1976; 25: 71
        • Hoff H.F.
        • Bradley W.A.
        • Heideman C.L.
        • Gaubatz J.W.
        • Karagas M.D.
        • Gotto Jr., A.M.
        Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions.
        Biochim. Biophys. Acta. 1979; 573: 361
        • Hollander W.
        Unified concept on the role of acid mucopolysaccharides and connective tissue proteins in the accumulation of lipids, lipoproteins and calcium in the atherosclerotic plaque.
        Exp. Mol. Path. 1976; 25: 106
        • Hollander W.
        • Paddock J.
        • Colombo M.
        Lipoproteins in human atherosclerotic vessels, Part 1 (Biochemical properties of arterial low density lipoproteins, very low density lipoproteins and high density lipoproteins).
        Exp. Mol. Path. 1979; 30: 144
        • Hollander W.
        • Colombo M.
        • Paddock J.
        Lipoproteins in human atherosclerotic vessels, Part 2 (Biochemical properties of the major apolipoproteins of arterial low density and very low density lipoproteins).
        Exp. Mol. Path. 1979; 30: 172
        • Havel R.J.
        • Eder H.A.
        • Bragdon J.H.
        Distribution and chemical composition of ultracentrifugally separated lipoproteins and chylomicrons.
        J. Clin. Invest. 1955; 34: 1345
      1. Hoff, H.F., Isolation and purification of lipoproteins containing apo B from human atherosclerotic plaques by affinity chromatography, in preparation.

        • Weber K.
        • Osborn M.
        The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis.
        J. Biol. Chem. 1969; 244: 4406
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        Protein measurement with the Folin phenol reagent.
        J. Biol. Chem. 1951; 193: 265
        • Roeschlau P.
        • Bernt E.
        • Gruber W.
        Enzymatische Bestimmung des Gesamt-Cholesterins im Serum.
        J. Clin. Chem. Clin. Biochem. 1974; 12: 403
        • Bartlett G.R.
        Phosphorous assay in column chromatography.
        J. Biol. Chem. 1959; 234: 466
        • Carlson L.A.
        Determination of serum triglycerides.
        J. Atheroscler. Res. 1963; 3: 334
        • Skipski P.
        • Barclay M.
        Thin-layer chromatography of lipids.
        in: Methods of Enzymology. Vol. 14. Academic Press, New York1969: 530
        • Morrison W.R.
        • Smith L.M.
        Preparation of fatty acid methyl esters and dimethyl-acetals from lipids with boron fluoride-methanol.
        J. Lipid Res. 1964; 5: 600
        • Hoff H.F.
        • Morrisett J.D.
        • Gotto Jr., A.M.
        Interaction of phosphatidylcholine and apolipoprotein-alanine—Electron microscopic studies.
        Biochim. Biophys. Acta. 1973; 296: 653
        • Scheidegger J.A.
        Une micro-méthode de l`immuno-électrophorese.
        Int. Arch. Allergy. 1955; 7: 103
        • Weeke B.
        Crossed immunoelectrophoresis.
        in: A Manual of Quantitative Immunoelectrophoresis. Universitets Forlaget, Oslo, Bergen, Tromsö1973: 47
        • Blumenkrantz H.
        • Asboe-Hansen G.
        An assay for total hexosamine and a differential assay for glucosamine and galactosamine.
        Clin. Biochim. 1976; 9: 269
        • Yamagata T.
        • Saito H.
        • Habuchi O.
        • Suzuki S.
        Purification and properties of bacterial chondroitinases and chondrosulfatases.
        J. Biol. Chem. 1968; 243: 1523
        • Kane J.P.
        • Hardman D.A.
        • Paulus H.E.
        Heterogeneity of apolipoprotein B-Isolation of a new species from human chylomicrons.
        in: Proc. Nat. Acad. Sci. (USA). 77. 1980: 2465
        • Nakamura M.
        • Torii S.
        • Yatsuki K.
        • Kikuchi Y.
        • Yamamoto H.
        Cerebral atherosclerosis in Japanese, Part 1 (Lipids and glycosaminoglycans in cerebral arteries).
        Atherosclerosis. 1971; 13: 185
        • Hoff H.F.
        • Gaubatz J.W.
        • Gotto Jr., A.M.
        Apo B concentration in the normal human aorta.
        Biochem. Biophys. Res. Comm. 1978; 85: 1424
        • Aulinskas T.H.
        • Van der Westhuyzen D.R.
        • Bierman E.L.
        • Gevers W.
        • Coetzee G.A.
        Retroendocytosis of low density lipoprotein by cultured bovine aortic smooth muscle cells.
        Biochim. Biophys. Acta. 1981; 664: 255
        • Fowler S.
        • Berberian P.A.
        • Shio H.
        • Goldfischer S.
        • Wolinsky H.
        Characterization of cell populations isolated from aortas of rhesus monkeys with experimental atherosclerosis.
        Circ. Res. 1980; 46: 520
        • Barber A.A.
        • Bernheim F.
        Lipid peroxidation-Its measurement, occurrence and significance in animal tissues.
        in: Advances in Gerontological Research. Vol. 2. Academic Press, New York1967: 355
        • Moncada S.
        • Amezcua J.L.
        Prostacyclin, thromboxane A2 interactions in haemostasis and thrombosis.
        Haemostasis. 1979; 8: 252
        • Lee D.M.
        Malondialdehyde formation in stored plasma.
        Biochem. Biophys. Res. Comm. 1980; 95: 1663
        • Hoff H.F.
        LDL with altered surface charge—A new risk factor in atherogenesis.
        Artery. 1979; 6: 178
        • Goldstein J.L.
        • Ho Y.K.
        • Basu S.K.
        • Brown M.S.
        Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.
        in: Proc. Nat. Acad. Sci. (USA). 76. 1979: 337
        • Brown M.S.
        • Basu S.K.
        • Falck J.R.
        • Ho Y.K.
        • Goldstein J.L.
        The scavenger cell pathway for lipoprotein degradation—Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages.
        J. Supramol. Struct. 1981; 13: 67
        • Mahley R.W.
        • Innerarity T.L.
        • Weisgraber K.H.
        • Oh S.Y.
        Altered metabolism (in vivo and in vitro) of plasma lipoproteins after selective chemical modification of lysine residues of the apoproteins.
        J. Clin. Invest. 1979; 64: 743
        • Fogelman A.M.
        • Schechter I.
        • Seager J.
        • Hokom M.
        • Child J.S.
        • Edwards P.A.
        Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation in human monocyte -macrophages.
        in: Proc. Nat. Acad. Sci. (USA). 77. 1980: 2214
        • Schechter I.
        • Fogelman A.M.
        • Haberland M.E.
        • Seaer J.
        • Hokom M.
        • Edwards P.A.
        The metabolites of native and malondialdehyde-altered low density lipoproteins by human monocytemacrophages.
        J. Lipid Res. 1981; 22: 63
        • Goldstein J.L.
        • Hoff H.F.
        • Ho Y.K.
        • Basu S.K.
        • Brown M.S.
        Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/cholesteryl esters.
        Arteriosclerosis. 1981; 1: 210
        • Shepherd J.
        • Packard C.J.
        • Grundy S.M.
        • Yeshurun D.
        • Gotto Jr., A.M.
        • Taunton O.D.
        Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man.
        J. Lipid Res. 1980; 21: 91
        • Small D.M.
        The physical state of lipids of biological importance—Cholesteryl esters, cholesterol, triglyceride.
        Adv. Exp. Med. Biol. 1970; 7: 55
        • Pattnaik N.M.
        • Zilversmit D.B.
        Interactions of cholesteryl ester exchange protein with human plasma lipoproteins and phospholipid vesicles.
        J. Biol. Chem. 1979; 254: 2782
        • Barter B.J.
        • Gooden J.M.
        • Rajaram O.V.
        Species differences in the activity of a serum triglyceride transferring factor.
        Atherosclerosis. 1979; 33: 165
        • Redgrave T.G.
        • Small D.M.
        Quantitation of the transfer of surface phospholipid of chylomicrons to the high density lipoprotein fraction during the catabolism of chylomicrons in the rat.
        J. Clin. Invest. 1979; 64: 165
        • Gaubatz J.W.
        • Heideman C.L.
        • Dahlen G.
        • Hoff H.F.
        Characterization of a lipoprotein containing apo B in LDL density range from human aorta.
        Circulation. 1980; 62: 861
        • Jurgens G.
        • Kostner G.M.
        Studies on the structure of the Lp(a) specific antigen.
        Immunogenetics. 1975; 1: 560
        • Ehnholm C.
        • Garoff H.
        • Renkonen O.
        • Simmons K.
        Protein and carbohydrate composition of Lp(a) lipoproteins from human plasma.
        Biochemistry. 1972; 11: 3229
        • Shen M.M.S.
        • Krauss R.M.
        • Lindgren F.T.
        • Forte T.M.
        Heterogeneity of serum low density lipoproteins in normal human subjects.
        J. Lipid Res. 1981; 22: 236