Lipoproteins size fractionation

Whole plasma can also be fractionated into specific lipoprotein size classes to further resolve the underlying biochemistry and metabolism of tissues that deliver these lipids to blood and selectively remove them. Thus, TrueMass analysis can be used to measure the lipid profiles of very-low-density lipoprotein, quantify the lipid pathways responsible for metabolic changes in the liver and measure profiles of high-density lipoprotein to quantify the flux of lipids in reverse cholesterol transport. 

Similar problems occur for the nephelometric and turbidimetric methods, where the sizes of the IgG-Lp(a) complexes depend upon that of apo(a) itself (L2, W4). Furthermore, problems due to interferences from elevated plasma triglyceride are commonly encountered in the precipitation techniques (C3). As Lp(a) can be redistributed among the Lp(a) fraction and the triglyceride-rich lipoproteins, especially in patients after a fatty meal (B11), these methods are not appropriate for monitoring Lp(a) levels and distribution in plasma. 

Chromatographic procedures have been applied increasingly in the fractionation and purification of plasma lipoproteins (Bll, L3, W2). Agarose media have proved to be particularly valuable because of their sieving properties for particles in the size range of plasma lipoproteins, including the low- and very low-density classes (SI). 

It thus seemed that the origin of the various components in meat volatiles could best be established by analyzing irradiation-induced compounds in meat protein and meat fat separately. Accordingly, a 500-gram sample of meat, the same size of sample normally used in irradiation studies of whole meat, was separated into a protein, a lipid, and a lipoprotein fraction by means of a methanol-chloroform extraction of the fat. The dry, air-free, fractions were then irradiated separately with 6 megarads of gamma radiation in the manner used for whole meat. The analytical results (Table V) show clearly that mainly sulfur compounds and aromatic hydrocarbons are formed in the protein fraction, whereas mainly aliphatic hydrocarbons are formed from the lipid. The lipoprotein fraction produced, as expected, both aliphatic hydrocarbons and sulfur compounds. Only the lipoprotein fraction had a characteristic irradiation odor. 

In normolipidemic subjects, apoE is found not only in HDLc-like particles but also in two other fractions associated with triglyceride-rich lipoproteins. These are VLDL, and a lipoprotein class intermediate in size between VLDL and LDL (G3). The latter may be the normal counterpart of the (3-VLDL which accumulates in Type III hyperlipoproteinemia and in cholesterol-fed animals. 

Lipoproteins which are difficult to investigate by techniques relying on the particle density as well as on the particle size (S-FFF, ultracentrifugation, etc.) can be successfully separated by Fl-FFF which yields the particle size distribution of high, low and very low density lipoproteins [437,438]. Furthermore, Fl-FFF has been used to fractionate ground minerals by size [439]. 

Fig. 6. Fractionation of proteins in 50 ml of human plasma on Sephadex G-200. Eluent, 0.1 M Tris + 1 M NaCI, pH 8.0 elution rate, 68 ml/hour column size, 7 X 50 cm absorption measured with LKB Uvicord recorder. Region A contains /32m-(19 S 7-) and asM-globulins, a- and /3-lipoproteins, and fibrinogen B, 7 S 7-globulin C, transferrin and D, albumin. (Flodin, 1962.)...

The quantitative relationship between cholesterol intake and cholesterol levels is still controversial, especially because in humans, there appears to be a high individual variability in processing of dietary cholesterol. However, numerous animal and human studies support the concept that dietary cholesterol can raise LDL-cholesterol levels and change the size and composition of these particles as well. LDL particles become larger in size and enriched in cholesterol esters. Mechanisms contributing to these events include an increase in hepatic synthesis of apoB-containing lipoproteins, increased conversion of VLDL remnants to LDL, or a decrease in the fractional catabolic rate for LDL. Reduced LDL receptor activity due to an increase in hepatic cholesterol content, secondary to excess dietary cholesterol, may lead to a decreased uptake of both LDL and VLDL remnants. 

For simplicity of calculation, the core was assumed to contain all of the triglyceride and cholesteryl ester, although it is known that small amounts of the core lipids are dissolved in the surface monolayer, where they represent about 3 mol% of the surface lipids, and a larger fraction, about one ninth of the cholesterol, is dissolved in the core (Miller and Small, 1987). The presence of core lipids in the lipoprotein surface is very important metabolically, for the lipases and transfer proteins have access to these core lipids without having to penetrate the surface monolayer. For the calculation of composition, density, and size, however, the effects of component transfer between surface and core affect these quantities about one part in the fourth significant figure, and have been neglected in Table II. 

Plasma lipoproteins, as macromolecular complexes that vaiy considerably in size, composition, and function, present considerable analytical challenge (see Table 26-4). For clinical purposes, lipoprotein concentrations have been traditionally expressed in terms of their cholesterol content, because they carry virtually all of the cholesterol that circulates in the plasma. This simplifies the methods used to measure lipoproteins because the fipoprotein fractions of interest have only to be separated from each other the other plasma proteins do not have to be removed. 

The molecular masses of proteins range up to 2,750 kDa for the beta-lipoproteins, and the dynamic ranges for the plasma proteins range from millimoles per liter for albumin to femtomoles for the proteins in lower abundance. The molecular size of the individual protein is an important factor in determining its distribution and transport by the active and passive mechanisms of the body. Some proteins move freely in the extracellular and intravascular spaces, while other intracellular proteins are released only after cell damage. Some of the major plasma proteins are listed in Table 8.1, together with the broad protein fractions designated by their simple electrophoretic mobilities. 

The lipoproteins are macromolecules with varying complexes of lipids where the hydrophobic lipid portions—cholesterol esters and triglycerides—are localized at the core of the molecules. The amphipathic surface layers surrounding the core contain the apolipoproteins and phospholipids. The lipoproteins vary in size, density, lipid composition, and apolipoprotein constituents, and they ean be classihed by size, the flotation rate determined by ultracentrifugation, or their electrophoretic mobilities. Put simply, the density of a lipoprotein particle is determined by the relative amounts of lipid and protein contained in the particle. Chylomicrons and very low density lipoproteins have the highest lipid content and the lowest protein content thus, very excessive amounts of chylomicrons float on the surface of plasma. In descending order of size, the broad lipoprotein fractions (with their electrophoretic mobility) are... 

These broad lipoprotein classes are heterogeneous, with particle fractions of differing size, density, and function the subclasses are denoted by subscript numbers (e.g., HDLj). 

Fig. 2. Light scattering intensity (LSI arbitrary units) of serum lipoproteins of different size after loading with monoglycerides (MG), free fatty acids (FFA), a 2 1 mixture of free fatty acids with monoglycerides or triglycerides (TG). All fats had the fatty acid composition of safflower seed oil the monoglycerides had equilibrium isomeric composition, and fats were fed at 1 g/kg body weight. Lipoproteins were separated by ultrafiltration into fractions of different size [diameter (d) > 400 nm, 100 < d < 400 nm, and d < 100 nm].

The detection of abnormal lipoproteins can be done more effectively from the elution patterns monitored by protein or choline-containing phospholipids for the isolated lipoprotein fraction by the ultracentrifugation. In Fig. 20, the elution patterns of choline-containing phospholipids for each ultracentrifugal fraction are presented for three subjects normal female and patients with acute hepatitis and lecithin cholesterol acyltransferase deficiency. In contrast to normal subjects, all lipoprotein fractions for two pathological cases show the existence of abnormal lipoproteins in which a correlation between the particle size and density dose not exist. 

Functions.—Heparin fractionated by gel filtration appeared to bind to two sites on antithrombin III (association constants 0.6 x 10 and 0.2 x 10 moll" ), whereas heparin prepared by affinity chromatography on matrix-bound antithrombin III appeared to bind to only one site (association constant 2.3 x 10 moll ). These results suggest that one of the binding sites on antithrombin III does not bind the most active heparin components, but accommodates heparin-like molecules which, although similar in size to the active heparin components, have little or no anticoagulant activity. Heparins with high or low affinities for antithrombin III exhibited no differences in their abilities to bind lipoprotein lipase. Studies of the interaction between the lipoprotein lipase from cow s milk and Sepharose-immobilized heparin have shown that heparin is poly-disperse. Whereas heparin facilitated complex formation between a-thrombin and antithrombin III, it had little effect on the interaction between p-thrombin and antithrombin III. ... 

The methods of X-ray and neutron small-angle scattering have disclosed important information on the molecular arrangement within lipoproteins which is not directly available from any other source. A crucial prerequisite for the successful application of these methods has been the preparation of well-defined fractions out of the broad spectrum of particle sizes and compositions naturally occurring in serum. Therefore, up to now, an analysis by these methods has been possible only for the subfractions of HDL and LDL which have proven to be sufficiently close to monodispersity. 

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