High-density lipoproteins separation

Lipoproteins are divided into five basic types. The largest particles are the chylomicrons, followed by the very low-density lipoproteins, intermediate lipoproteins, low-density proteins, and finally the smallest— high-density lipoproteins. Separation of the aforementioned cholesterol-containing complexes is accomplished by ultracentrifugation. 

Apolipoproteins ( apo designates the protein in its lipid-free form) combine with lipids to form several classes of lipoprotein particles, spherical complexes with hydrophobic lipids in the core and hydrophilic amino acid side chains at the surface (Fig. 21-39a). Different combinations of lipids and proteins produce particles of different densities, ranging from chylomicrons to high-density lipoproteins. These particles can be separated by ultracentrifugation (Table 21-2) and visualized by electron microscopy (Fig. 21-39b). 

The small particles of plasma lipoprotein, which carry triacylglycerols, can be separated according to their buoyant densities by centrifugation. They have been classified into five groups of increasing density but smaller size as chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density (LDL), and high density lipoproteins (HDL) (Table 21-1 and Fig. 21-2). Each lipoprotein particle contains one or more apolipoproteins (Table 21-2), whose sizes vary from the enormous 4536-residue apoB-100 to apoC-II and apoC-III, each of which contains just 79 residues73 and the 57-residue apoC-I.7b... 

Disorders in lipoprotein metabolism are critical in the etiology of several disease states, such as coronary heart disease and atherosclerosis. Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. A simple chromatographic method for the separation of high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) from intact serum or plasma has been reported recently [65]. The separation was achieved by using an hydroxyapatite column and elution with pH 7.4 phosphate buffer with lOOpl injections of whole... 

Plasma lipoproteins are generally classified by their density and separation achieved with ultracentrifugation. According to this density-based classification system, the major lipoprotein classes are chylomicrons (CH), very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). 

M., A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor. J, Lipid Res. 23, 1342-1353 (1982). 

K21. Kostner, G. M., and Holasek, A., The separation of human serum high density lipoproteins by hydroxyapatite column chromatography. Biochim. Biophys. Acta 488, 417-431 (1977). 

M22. Malmendier, C. L., and Ameryckx, J. P., S-peptides—new apoproteins of human high density lipoproteins induced by glucose infusions. Clin. Chim. Acta 111, 267-270 (1981). M23. Malmendier, C. L., Christophe, J., and Ameryckx, J. P., Separation and partial characterization of new apoproteins from human plasma high density lipoproteins. Clin. Chim. Acta 99, 167-176 (1979). 

The various lipoproteins found in plasma can be separated by ultraccntrifugal techniques into chylomicrons, vcry-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). These correlate with the electrophoretic separations of the lipoproteins as follows chylomicrons, pre-/3-lipoprotcin (VLDL). broad )3-lipoprotein (IDL). /3-lipoprotein (LDL). and ar-lipoprotcin (HDL). 

Lippi U, Graziani MS, Manzato F, SchineUa M. Procedure for effective separation of high-density lipoproteins in normal serum and hypertriglyceri-demic samples. Clin Biochem 1987 20 313-15. 

The assay used by Pattnaik et al. (1978) to isolate the cholesteryl ester transfer protein in human plasma measured the transfer of 3H-labeled cholesteryl ester from low density to high density lipoprotein. The transfer reaction was terminated by adding MnC and centrifuging to sediment the low density lipoprotein. A heparin, MnCl2 precipitation step may also be used however, in the presence of a phosphate buffer, heparin is not necessary for precipitation of low density lipoprotein. Routine assays were found to be reproducible to within 10%. The increase in high density lipoprotein radioactivity is linear with time until about 25% of the initial low density lipoprotein radioactivity is transferred. The lipoproteins may also be separated by ultracentrifugation. When these two methods were compared, the results agreed within 10%. The precipitation technique is much faster (less than 15 min compared with 18 hr) and simpler. 

Ellsworth et al. (1982) used a small heparin-Sepharose column to separate high density lipoprotein from low density and very low density lipoprotein by adjusting the concentration of NaCl and SDS in the eluting buffer. The results obtained by this method were the same as those gained by ultracentrifugation. 

Bernardi G. and Cook W.H. (1960b). Separation and characterization of the two high-density lipoproteins of egg yolk, a- and /3-lipovitellin. Biochim. Biophys. Acta 44 96-105. 

Lipoprotein nomenciature is based on mode of separation. When preparative uitracentrifugation is used, lipoproteins are separated according to their density and identified as very-iow-density iipoproteins (VLDLs), intermediate-density lipoproteins (IDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). When electrophoresis is employed in the separation, lipoproteins are designated as pre-(3, (3, and a. The IDLs are mainly found in the pre-(3 fraction as a second electrophoretic band and are currently... 

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