Lipoproteins ultracentrifugation

Serum Lipoproteins, Ultracentrifugal Analysis of (de Lalla and Gofman) 1 459... 

The lipoproteins can be separated by different means paper electrophoresis (main fractions a- and /J-lipoproteins), ultracentrifugation (low- and high-density fractions), see for instance Fasoli et al. (1956) and Lindgren and Gofman (1956) or Cohn s el al. (1950), microfractionatingmethod. 

Lipoproteins (from human plasma). Individual human plasma lipid peaks were removed from plasma by ultracentrifugation, then separated and purified by agarose-column chromatography. Fractions were characterised immunologically, chemically, electrophoretically and by electron microscopy. [Rudel et al. Biochem J 13 89 1974.]... 

Lp(a), moving, like very low-density lipoproteins (VLDL), in the pre-(3,-lipoprotein fraction upon electrophoresis (B8, B9), but not floating like VLDL in ultracentrifugation (H29), partly resembles low-density lipoprotein (LDL). Its protein moiety consists of one glycoprotein molecule, named apolipoprotein (a)... 

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). 

Mills, G. L., Seidel, D., and Alaupovic, P., Ultracentrifugal characterization of a lipoprotein occurring in obstructive jaundice. Clin. Chem. Acta 26, 239-244 (1969). Nelson, G., ed., Blood Lipids and Lipoproteins Quantification, Composition and Metabolism, 980 pp. Wiley (Interscience), New York, 1972. 

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. 

Ultracentrifugation Minimal non-specific binding and osmotic volume shifts. Large plasma volumes required, long assay time, issues such as sedimentation, back diffusion and viscosity. Potential for lipoprotein contamination of plasma water layer. [28, 29]... 

Ultracentrifugation of fasted serum leads to the separation of the VLDL lipoproteins from the LDL and the HDL lipoproteins. The cholesterol concentration is then measured in the VLDL fraction. 

Hulley SB, Cook SG, Wilson WS, Nichaman MZ, Hatch FT, Lindgren FT (1971) Quantitation of serum lipoproteins by electrophoresis on agarose gel standardization in lipoprotein concentration units (mg-100 ml) by comparison with analytical ultracentrifugation. J Lipid Res 12 420-433... 

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). 

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... 

Figure 1. Pedigree pattern of the B family. Relationship of the proband (T.B.) with the clinical phenotype of homozygous familial hypercholesterolemia to her other relatives whom we studied is shown. Lipoprotein patterns were determined after ultracentrifugation using NIH outpoints (51). F indicates that fibroblast cell lines were established from skin biopsies. Males, H Females, O.

Human plasma lipoproteins and lipoprotein deficient plasma (LPDS) were prepared from normal plasma of healthy individuals by differential ultracentrifugation on KBr gradients (16). 

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). 

For lipids, authors are far from agreement and it is difficult to compare the values reported. A selection of published figures is, however, given in Table 11. Comparison between electrophoretic and ultracentrifuge fractions seems an interesting field (P12, P13, P14, P15). Two-dimensional techniques are modifying our concepts on lipoproteins and give consistent results, as will be seen at the end of Part II. 

Lipoproteins interact with enzymes (e.g., lecithin cholesterol acyltrans-ferase, or lipases), with lipid transfer proteins, or with receptors on cell surfaces. The composition of a lipoprotein class depends upon the results of these kinds of interactions. Ultracentrifugation itself may result in minor changes in lipoprotein composition (see Sections 6 and 6.1). 

---