The association of very-low-density lipoprotein (VLDL) with atherosclerosis remains controversial. However, studies have shown that oxidative modification of VLDL can promote foam cell formation, leading to the development of atherosclerosis. A rapid method is described which will allow the significance of VLDL oxidation to be assessed in clinical studies. VLDL was isolated from heparinized plasma by a 1-h, single spin ultracentrifugation. Total protein was standardized to 25 mg/L. Oxidation was promoted by the addition of copper ions (17.5 μmol/L, final concentration) incubated at 37°C. Conjugated diene production was followed at 234 nm. Total assay preparation time was 2 h. Urate greatly inhibited the oxidation of VLDL and was successfully removed by size exclusion chromatography. VLDL isolated from frozen plasma (−70°C) was stable for 15 weeks. This simple, rapid method for the isolation of VLDL may be applied to assess the significance of VLDL oxidation in disease.
WitztumJLSteinbergD.Role of oxidised low density lipoprotein in atherogenesis. J Clin Invest1991; 88: 1785–92.
2.
SteinbergDParthasarathySCarewTEKhooJCWitztumJL. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med1989; 320: 915–24.
EsterbauerHGebickiJPuhlHHJurgensG.The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med1992; 13: 341–90.
5.
ParthasarathySQuinnMTDawnCSchwenkeDCCarewTESteinbergD.Oxidative modification of beta-very low density lipoprotein. Potential role in monocyte recruitment and foam cell formation. Arterioscler Thromb Vasc Biol1989; 9: 398–404.
6.
HorriganSCampellJHCampellGR. Oxidation of beta-very-low-density lipoprotein by endothelial cells enhances its metabolism by smooth muscle cells. Arterioscler Thromb Vasc Biol1991; 11: 279–89.
7.
MorelDWChisolmGM. Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity. J Lipid Res1989; 30: 1827–34.
8.
KeidarSKaplanMRosenblatMBrookGJAviramM.Apolipoprotein E and lipoprotein lipase reduce macrophage degradation of oxidised very-low-density lipoprotein (VLDL), but increase cellular degradation of native VLDL. Metabol1992; 41: 1185–92.
9.
MohrDStockerR.Radical-mediated oxidation of isolated human very-low-density lipoprotein. Arterioscler Thromb Vasc Biol1994; 14: 1186–92.
10.
SwainJGutteridgeJMC. Pro-oxidant iron and copper, with ferroxidase and xanthine-oxidase activities in human atherosclerotic material. FEBS Lett1995; 368: 513–5.
11.
ChapmanMJGoldsteinSLagrangeDLaplaudPM. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J Lipid Res1981; 22: 339–58.
12.
LeonhartWPietzschJNitzscheS.Very-fast ultracentrifugation of human plasma lipoproteins: Influence of the centrifugal field on lipoprotein composition. Clin Chem Acta1994; 224: 21–32.
13.
CathcartSDominiczakMH. The measurement of lipoprotein subfractions in plasma using a tabletop ultracentrifuge. Ann Clin Biochem1990; 27: 459–64.
14.
The Lipid Research Clinics Program. The Lipid Research Clinics coronary primary prevention trial results II. The relationship of reduction in incidence of coronary heart disease. JAMA1984; 251: 365–75.
15.
ManciniGCarbonaraAOHeremansJF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem1965; 2: 235–54.
16.
BeckerW.Determination of antisera titres using the single radical immunodiffusion method. Immunochem1969; 6: 539–46.
17.
McDowellIFWMcEnenyJTrimbleER. A rapid method for measurement of the susceptibility to oxidation of low-density lipoprotein. Ann Clin Biochem1995; 32: 167–74.
18.
HalliwellB.Albumin—an important extracellular antioxidant. Biochem Pharmacol1988; 37: 569–71.
19.
FreiBStockerRAmesBN. Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci1988; 85: 9748–52.
20.
MayYSStoneWLLeclairIO. The effects of vitamin C and urate on the oxidation kinetics of human low density lipoprotein. Proc Soc Exp Biol1994; 206: 53–9.
21.
ChevionSBerryEMKitrosskyNKohenR.Evaluation of plasma low molecular weight antioxidant capacity by cyclic voltammetry. Free Rad Biol Med1997; 22: 411–21.
22.
JialalIVegaGLGrundySM. Physiologic levels of ascorbate inhibit the oxidative modification of low density lipoprotein. Atherosclerosis1990; 82: 185–91.
23.
VuillemierJPKeckE.Fluorometric assay of vitamin C in biological materials using a centrifugal analyser with fluorescence attachment. J Micronut Analysis1989; 5: 25–35.
24.
WolffSP. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Meth Enzymol1994; 233: 182–9.
25.
Naurooz-ZadehJTajaddini-SarmadiJWolffSP. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation—xylenol orange assay in conjunction with triphenylphosphine. Anal Biochem1994; 220: 403–9.
26.
McEnenyJLoughreyCMMcNameePTTrimbleERYoungIS. Susceptibility of VLDL to oxidation in patients on regular haemodialysis. Atherosclerosis1997; 129: 215–20.
27.
HeineckeJWRosenHChaitA.Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest1984; 74: 1890–4.
28.
KleinveldHAHak-LammersHLMStalenhoefAFHDemackerPNM. Improved measurement of low-density-lipoprotein susceptibility to copper-induced oxidation: Application of a short procedure for isolating low-density lipoprotein. Clin Chem1992; 38: 2066–72.
29.
RamosPGiesegSPSchusterBEsterbauerH.Effect of temperature and phase transition on oxidation resistance of low density lipoprotein. J Lipid Res1995; 36: 2113–28.
30.
GiesegSPEsterbauerH.Low density is saturable by pro-oxidant copper. FEBS1994; 343: 188–94.
31.
ZiouzenkovaOGiesegSPRamosPEsterbauerH.Factors affecting resistance of low density lipoproteins to oxidation. Lipids1996; 31: S71–6.
32.
PrincenHMGvan PoppelGVogelezangCBuytenhekRKokFJ. Supplementation with vitamin E but not β-carotene in vivo protects low density lipoprotein from lipid peroxidation in vitro. Effect of cigarette smoking. Arterioscler Thromb Vasc Biol1992; 12: 554–62.
33.
LambDJLeakeDS. The effect of EDTA on the oxidation of low density lipoprotein. Atherosclerosis1992; 94: 35–42.
