Isolation of human serum HDL, by zonal ultracentrifugation

High density lipoprotein subfraction-1 (HDLI) is thought to interact with the high-affinity apoprotein B, E receptors of peripheral cells and may act as a modulator of LDL binding and uptake. In the present study the concentration and composition of HDLl in normal and hypercholesterolemic sera were studied using zonal ultracentrifugation. To permit separation of the HDLl from VLDL, LDL, and Lp(a), the apoBcontaining lipoproteins were first precipitated from serum using the phosphotungstic acid/magnesium chloride (PTA/MgC12) method after which the supernatant fraction was subjected to zonal ultracentrifugation. It could be demonstrated that following PTA/MgC12 precipitation HDLl floats as a single peak at d 1.08-1.09 g/ml (NaBr) and is sufficiently separated from high density lipoprotein-2 (HDL2) and high density lipoprotein-3 (HDL3). The HDL2/HDL3 subfraction pattern was not affected by the precipitation method. As previously described, in vitro incubation of serum leads to the LCAT-dependent interconversion of HDL3 or HDLz. Using the technique described here, it was discovered that a simultaneous elevation of HDLl occurred. This increase in HDLl concentration could not be observed when LCAT was inhibited by heat inactivation or addition of Ellman's reagent. In normal fresh serum only a small HDLl peak could be detected, but in patients with familial hypercholesterolemia (apoB, E receptor deficiency) HDLl was elevated five to tenfold compared to normal values and further increased in concentration upon incubation of serum. On the other hand, in sera of patients with familial HDL deficiency (Tangier disease), HDLl was undetectable. Analysis of the HDL fractions in serum of a patient with abetalipoproteinemia revealed that following in vitro incubation there was formation of HDLl despite the lack of apoprotein B-containing 1ipoproteins.l These data support the concept that HDL1 formation occurs during LCAT-mediated HDL,/ HDLz interconversion in vitro.-Schmitz, G., and G. Assmann. Isolation of human serum HDLl by zonal ultracentrifugation. 3. Lipid Res. 1982. 23 903-910. Supplementary key words LCAT HDL2 HDL3 apoE Despite present understanding of the role of various lipoproteins in transporting cholesterol in blood, little is known about the factors that regulate the flux of cholesterol to and from the peripheral tissues. The homeostatic control of these two pathways may be critical in restricting cholesterol accumulation in tissues and thus of importance in preventing atherosclerosis. The bulk of the plasma cholesterol is transported in the LDL fraction. LDL transfers cholesterol to the peripheral tissues, where it is recognized by apoB, E receptor cells and, after cellular uptake, is degraded (1). Transfer from peripheral tissues to the liver is thought, on the other hand, to be mediated by high density lipoproteins (2, 3). Thus, the deposition of cholesterol in tissues theoretically may result from enhanced transfer by LDL or from a failure to eliminate cellular cholesterol via the H D L pathway. The precise mechanism by which H D L removes cholesterol from peripheral cells is not yet known. In addition to the possible role of circulating HDL3 as a cholesterol-acceptor macromolecule, tissue cholesterol may be alternatively removed by H D L precursors that are interconverted to HDL2 by the activity of 1ecithin:cholesterol acyltransferase (LCAT) (4). These particles may then be taken up by the liver as intact particles (5), or the cholesterol ester moiety may be transferred to the hepatic cells with subsequent reconstitution to form an HDL3 particle (6). A further H D L subfraction potentially involved in the transport of cholesterol is the apoprotein E-containing HDLl fraction. The present study provides evidence that HDLl is formed during the LCAT-mediated HDL3/HDL2 interconversion and that in the serum of patients with apoB, E receptor deficiency, HDLl is increased in concentration. It is suggested that the concentration of HDLl may be physiologically important in regulating LDL uptake by apoB, E receptor cells. MATERIAL AND METHODS