Chylomicrons ultracentrifugation

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). 

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). 

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). 

These lipids are insoluble in water and are classified on the basis of their ultracentrifugal properties into chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in order of ascending density. Table 2.4 gives the classification and roles of lipoproteins. 

Heparin-MnCb was selected as the precipitation reagent primarily for historical reasons because it was the method most commonly used in early studies to estabfish the relationship between HDL cholesterol concentration and the risk for CHD, The ultracentrifugation step was included to prevent interference with sedimentation of the apo B-lOO-containing fipoproteins by the lighter triglyceride-rich lipoproteins, VLDL, and chylomicrons. 

The CDC has also defined a reference method for LDL cholesterol based on the same techniques described above for HDL cholesterol. After ultracentrifugation to remove the VLDL and any chylomicrons present, the bottom fraction (d > 1.006) is subjected to precipitation by heparin and manganese as described previously. After measurement of cholesterol in the d > 1.006 fraction and in the heparin-Mn supernatant solution, LDL cholesterol is calculated by difference. It should be noted that the LDL fraction as measured by this reference method is a so-called broad-cut fraction including any IDL and Lp(a). 

LDL cholesterol measured in this way is unaffected by the presence of either chylomicrons or other triglyceride-rich lipoproteins, or by 3-VLDL. VLDL cholesterol is usually calculated from equation (18) rather than measured directly in the ultracentrifugal supernatant because it can be difficult to recover this fraction quantitatively, particularly when triglyceride concentrations are high. 

Intermediate-Density (Remnant) Lipoproteins Remnant lipoproteins include the lipolytic products of catabolism of the triglyceride-rich lipoproteins, VLDL and chylomicrons, occurring in the VLDL and LDL ranges. A traditionally defined fraction at the lighter end of the LDL density range, the IDL portion comprises the 1.006 to 1.019 g/mL fraction, which is obtained by sequential ultracentrifugation for quantitation, generally in terms of cholesterol content. 

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... 

To prepare pHJvitamin A-labeled chylomicrons (Green et ai, 1993), [ Hjretinol or retinyl acetate is administered intraduodenally to thoracic lymph duct-cannulated donor rats and lymph is collected at 4°C. Chylomicrons containing mainly pHJretinyl esters can be isolated from lymph by preparative ultracentrifugation for administration to recipient rats. Alternatively, aliquots of whole lymph can be injected to minimize handling of the dose (see below). Then the proportions of total lymph radioactivity and vitamin A mass in chylomicrons (typically >8590%) can be determined analytically. In either case, lymph preparations should be used for in vivo studies within 12 days of collection. 

The total lipoprotein fraction (d<1.210) prepared from serum by the ultracentrifugation was analyzed in the same HPLC conditions as in Fig. 7. As presented in Fig. 8, well separated peaks corresponding to chylomicrons+VLDL, LDL, HDL2 and HDLj are observed by monitoring at A2gQ. The elution volume of each lipoprotein peak in Fig. 8 is consistent with that of the reference standards in Fig. 7. The concentration of total cholesterol, triglycerides and phospholipids in each ml of eluate are plotted in the same figure. For all lipoprotein classes, the peak position of protein is consistent with those of the three lipid components. 

Although apoprotein A-IV exhibits the properties of an apolipoprotein [2], and recent data on its sequence [11, 12, 21] have shown that it contains 14.5 tandemly repeated docosapeptides that possess the potential to form amphipathic a-helices [11], it is mainly found unassociated with lipoproteins in human plasma [4,18,19,36]. The Apo A-IV fraction in the lipoprotein-free plasma compartment is still able to bind lipids, as shown by Weinberg and Scanu [37], who were able to reassociate Apo A-IV from the d = 1.21 g/ml infranate to a phospholipid-triglyceride emulsion. After reassociation Apo A-IV could be isolated by flotation in chylomicron-like particles upon ultracentrifugation. 

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