Density Lipoprotein Isolation

2 In order to minimize deterioration, it is recommended that all salt solutions used for lipoprotein fractionation contain 10 mg/100 ml of the disodium salt of ethylene-diaminetetraacetic acid (EDTA). 

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Hazen SL, Heinecke JW (1997) 3-Chlorotyrosine, a Specific Marker of Myeloperoxidase-Catalyzed Oxidation, Is Markedly Elevated in Low Density Lipoprotein Isolated from Human Atherosclerotic Intima. J Clin Invest 99 2075... 

Suenram, A., McConathy, W. J., and Alaupovic, P., Evidence for the lipoprotein heterogeneity of human plasma high density lipoproteins isolated by three different procedures. Lipids 14, 505-510 (1979). 

H13. Hazen, S. L., and Heinecke, J. W., 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075—2081 (1997). 

Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM, Pennathur S, Heinecke JW. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem 1997 272 3520-3526. 

Figure 13.15. Headspace gas chromatograms of copper-oxidized low density lipoprotein isolated from a, unsupplemented subject, and b, fish oil supplemented subject. From Frankel et al (1994), with permission from the AOCS Press.

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

Soine PJ, Blanke RV, Chinchilli VM, et al. 1984a. High-density lipoproteins decrease the biliary concentration of chlordecone in isolated perfused pig liver. J Toxicol Environ Health 14(2-3) 319-335. 

For the convenience of the reader, we have outlined the method of sequential flotation employed in our laboratory for separating chylomicrons VLDL, LDL, HDLa, HDLs, VHDL, and d> 1.25 bottom (Table 1). This method, the result of years of experience, has been highly reproducible in terms of the normal human population examined in this laboratory. Such a method may not necessarily apply to dyslipoproteinemic states, where modifications may be necessary, depending on the type of abnormality under consideration. It should also be stressed that any lipoprotein isolated is in need of purification this may be achieved by ultracentrifugation based on the assumption that contaminants are in loose association with the main complex. Whenever this purification is not achieved, other methods may be used as outlined below. For a discussion of the application of density gradient ultracentrifugation to the study of plasma lipoproteins, the reader is referred to a recent review (L3). 

Seidel, D., Alaupovic, P., Furman, R. H., and McConathy, W. J., A lipoprotein characterizing obstructive jaundice. II. Isolation and partial characterization of the protein moieties of low-density lipoproteins. J. Clin. Invest. 49, 2396-2407 (1970). 

Various attempts have been made to measure the formation of peroxides in isolated proteins and low density lipoproteins upon exposure to various oxidizing agents including ionizing radiation, transition metals involved in Fenton reaction, peroxyl radicals, photosensitizers and enzymatic oxidative systems (for reviews see References 195, 234 and 241). 

Buckley, C, Bund SJ, McTaggart F et al. Oxidized low-density lipoproteins inhibit endothelium-dependent relaxations in isolated large and small rabbit coronary arteries. J. Auton. Pharmacol. 16,... 

Terminalia catappa, Combretaceae, commonly used in the folk medicine in Taiwan, contains several C-glycosylflavones, two of them galloyl esters of vitexin and isovitexin. The isolated compounds were tested for antioxidative activity on Cu " /02-induced low-density lipoprotein peroxidation. IC50 values were 2.1 pM for vitexin derivatives and 4.5 pM for isovitexin derivatives compared to 4.0 pM for probucol chosen as positive control. 

Chacko, G. K. (1985). Modification of high density lipoprotein (HDL3) with tetranitro-methane and the effect on its binding to isolated rat liver plasma membranes.). Lipid Res. 26, 745-754. 

The third, and perhaps least understood, mechanism regulating contact pheromone production involves its transport to the cuticular surface. The detection of large amounts of hydrocarbons and pheromone internally, within the hemolymph, prompted an examination of lipid transport in B. germanica. Gu et al. (1995) and Sevala etal. (1997) isolated and purified a high density lipoprotein, lipophorin, that carries hydrocarbons, contact pheromone, and JH within the hemolymph. The accumulated evidence supports the idea that the hydrocarbons and contact pheromone components are produced by oenocytes within the abdominal integument, carried by lipophorin, and differentially deposited in the cuticle and ovaries (Fan et al.,... 

To test this hypothesis, very low density lipoprotein (VLDL, d<1.0 gm/ml), low density lipoprotein (LDL, d=l.02-1.063) and high density lipoprotein (HDL, d=l.09-1.21) were isolated from outdated human plasma by ultracentrifugation according to established procedures (27,28), using potassium bromide for density adjustments and stored at -20° C in the presence of 20% sucrose before use. The purity of individual lipoprotein fractions thus obtained was established by polyacrylamide gel electrophoresis in sodium dodecyl buffer system (2 ) and filtration through a Sepha-rose 6B column, equilibrated with 0.2 M potassium bromide in 0.1 M sodium phosphate buffer, pH 7.2. Protein (30) and cholesterol... 

A27. Assmann, G., Herbert, P. N., Fredrickson, D. S., and Forte, T., Isolation and characterization of an abnormal high density lipoprotein in Tangier disease. ]. Clin. Incest. 60, 242-252 (1977). 

B43. Brewer, H. B., Jr., Fairwell, T., Larue, A., Ronan, R., Houser, A., and Bronzert, T., The amino acid sequence of human apoA-1, an apolipoprotein isolated from high density lipoproteins. Biochem. Biophys. Res. Common. 80, 623-630 (1978). 

E8. Eriksen, N., and Benditt, E. P., Isolation and characterization of the amyloid-related apoprotein (SAA) from human high density lipoprotein. Proc. Natl. Acad. Sci. U.S.A. 77, 6860-6864 (1980). 

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