The isolation of lipoproteins from human plasma by ultracentrifugation in zonal rotors

The major classes of lipoproteins were isolated from human plasma by ultracentrifugation in continuous density gradients using the Ti-14 and Ti-15 zonal rotors. Chylomicrons + VLDL, LDL, and HDL were separated from each other and from the more dense residual proteins (albumin fraction) of plasma by rate-zonal flotation in NaBr gradients in the density range 1.0-1.4. The chylomicron-VLDL fraction was subfractionated into constituent chylomicrons and VLDL by zonal ultracentrifugation in NaBr gradients in the density range 1.0-1.1. Plasma lipoproteins were analyzed for composition of lipids and content of protein, for electrophoretic mobility on paper, and for antigenic determinants by immunoelectrophoresis and immunodiffusion. Flotation constants (S(f)) of the LDL and HDL were calculated from measurements made in the analytical ultracentrifuge. Lipoproteins isolated from plasma by zonal ultracentrifugation were identical by these criteria to lipoproteins isolated by the usual procedure of sequential ultracentrifugation in solvents of increasing density. The procedure of zonal ultracentrifugation is rapid, quantitative, and less laborious than sequential techniques. Lipoproteins isolated by zonal ultracentrifugation are relatively uncontaminated by other proteins and extensive washing is therefore unnecessary. Zonal ultracentrifugation is more than a preparative method for the plasma lipoproteins; it is also an analytical procedure in that a record is obtained of the distribution and quantity of the lipoprotein within the continuous density gradient.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation.

Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.     

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.     

Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta).

A group of 14 adult male rhesus monkeys was maintained on a low cholesterol-high fat diet. Periodically, animals were fasted and blood samples were taken for characterization of the plasma lipoproteins. Complete separation of individual plasma lipoprotein classes was not achieved by traditional sequential ultracentrifugation techniques. Rather, initial separation of lipoprotein classes according to size was effected and density centrifugation was used subsequently for further separation. At least six lipoprotein fractions were identified, each of which was unique as defined by the properties of size, density (d), and electrophoretic mobility. These lipoprotein fractions were characterized by determination of chemical compositions and apoprotein patterns. The lipoproteins present in highest concentration in these monkeys were designated as region IV lipoproteins. This fraction had alpha-migration on agarose electrophoresis, 1.063 < d < 1.225, and the size, composition, and apoprotein pattern characteristic of HDL. No fewer than three fractions were identified with densities that overlapped the 1.019 < d < 1.063 range. Of these, the fraction designated as region III lipoproteins was present in highest concentration, had beta-migration by agarose electrophoresis, a predominant B apoprotein, and a chemical composition and size characteristic of LDL. Two larger subfractions, identified as region II lipoproteins, were separated from each other at a density of 1.050 g/ml. Agarose electrophoresis showed that the fraction with d < 1.050 had a migration intermediate between beta and pre-beta. The chemical composition and apoprotein pattern were consistent with the possibility that these lipoproteins were remnants of VLDL catabolism. The fraction with d > 1.050, had pre-beta mobility and a size and composition similar to the Lp(a) lipoprotein in plasma of human beings. At least two VLDL subfractions, identified as region I and IIa lipoproteins, were found although both were present in very low concentrations. Region I lipoproteins were larger and contained relatively more cholesteryl ester and more of the apoproteins that migrated with the mobility of apo-B and arg-rich apoprotein in SDS-polyacrylamide gel electrophoresis. Some of the region I lipoproteins were beta-migrating by agarose electrophoresis. These results suggested the possibility that a beta-migrating VLDL was present in these normal animals.     

Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100.

A monoclonal antibody to apolipoprotein (apo) B-100 (JI-H) with unique binding properties has been used to separate a population of triglyceride-rich lipoproteins from blood plasma of normotriglyceridemic individuals and patients with various forms of hypertriglyceridemia. This antibody fails to recognize an apoE-rich population of very low density lipoproteins (VLDL) containing apoB-100 as well as all triglyceride-rich lipoproteins containing apoB-48, but it binds other VLDL that contain apoE and almost all lipoproteins that contain apoB-100, but no apoE. The unbound triglyceride-rich lipoproteins separated by ultracentrifugation after separation from plasma by immunoaffinity chromatography contained 10-13% of the apoB of triglyceride-rich lipoproteins from three normotriglyceridemic individuals, 10-29% of that from five patients with endogenous hypertriglyceridemia, 40-48% of that from three patients with familial dysbetablipoproteinemia, and 65% of that from a patient with lipoprotein lipase deficiency. In all cases, the unbound triglyceride-rich lipoproteins contained more molecules of apoE and cholesteryl esters per particle than those that were bound to monoclonal antibody JI-H, and they were generally depleted of C apolipoproteins. These properties resemble those described for partially catabolized remnants of chylomicrons and VLDL. The affinity of the unbound lipoproteins for the low density lipoprotein (LDL) receptor varied widely, and closely resembled that of the total triglyceride-rich lipoproteins from individual subjects. Our results demonstrate that remnant-like chylomicrons and a population of remnant-like VLDL can be isolated and quantified in blood plasma obtained in the postabsorptive state from normotriglyceridemic and hypertriglyceridemic individuals alike.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

Isolation of plasma lipoproteins by zonal ultracentrifugation in the B14 and B15 titanium rotors

Lipoproteins were isolated from plasma of man, dog, rabbit, rat, and chicken by ultracentrifugation in continuous density gradients using the B14 titanium and B15 titanium zonal rotors. Both the VLDL and the LDL of human plasma were separated easily from the HDL and from the other more plentiful plasma proteins by centrifugation for only 1 or 2 hr in the B14 or B15 rotor, respectively. Satisfactory separation of the HDL from the more dense plasma proteins was not achieved with these rotors. The human LDL achieved isopycnic equilibrium (d 1.04) on prolonged periods (> 24 hr) of centrifugation in a sucrose-KBr density gradient. The pattern of distribution of cholesterol and phospholipid throughout the density gradient coincided with the pattern of distribution of the lipoprotein-protein measured spectrophotometrically or chemically. The concentration of cholesterol and phospholipid in the lipoproteins isolated by zonal ultracentrifugation agreed with analyses reported for lipoproteins isolated by sequential centrifugation in solutions of increasing density. The lipoproteins isolated by zonal ultracentrifugation were characterized further by their electrophoretic behavior. The fractions which were identified as the LDL (d 1.04-1.05) from all species migrated on paper as a beta-globulin; the LDL from plasma of dogs contained an additional component which has been designated as an alpha(2)-globulin. The fractions which were identified as the HDL from all species migrated as an alpha(1)-globulin. Reaction of human LDL with either rabbit antihuman beta-lipoprotein or rabbit antihuman serum resulted in a single immunodiffusion band. The S(f, 1.063) of the human LDL was calculated to be 6.0. When plasma from humans or rabbits was centrifuged in the B15 rotor, the HDL was not visible as a distinct peak and was not separable from the bulk of the more dense plasma proteins; when plasma from dogs or chickens was centrifuged under identical conditions, the HDL was clearly detectable. Even though the mean density of the HDL from dogs or chickens was not different from that of man or rabbits, the visibility of this lipoprotein in dogs and chickens was probably due to its high concentration in the plasma of these species. When plasma from the rat was centrifuged under similar conditions, the HDL was also clearly in evidence. Although rat plasma contained a relatively small concentration of HDL, the lipoprotein had a lower mean density than did the HDL of the other species and was therefore more easily separable from the dense plasma proteins. The procedure of zonal ultracentrifugation for the isolation of lipoproteins by flotation is simultaneously preparative and analytical and should find useful application in the investigation of the soluble lipoproteins from plasma and tissues.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.     

Interaction of cremophor EL with human plasma

1. Interaction of cremophor EL (CRM) with human plasma lipoproteins and nonlipoproteins has been investigated by ultracentrifugation. 2. VLDL has only a low or negligible capacity to bind CRM, i.e. there is little or no change in the optical absorption at 280 nm of VLDL when CRM is added. 3. A low density subfraction of low density lipoproteins seems to associate substantially with CRM at relatively low CRM concentrations (1-3 mg/ml), but such association is not evident for CRM concentrations in the region 12-116 mg/ml. 4. Low density lipoproteins (LDL) may act as a carrier for CRM-emulsions, yet there seems to be no concomitant change in the 280 nm optical absorption of the proteins of LDL. 5. The position in the gradient (i.e. in the centrifugation tube after centrifugation) of high density lipoproteins (HDL) is shifted towards lower density in the presence of 1-4 mg CRM/ml. For higher concentrations of CRM, a destruction of HDL can be observed: the HDL distribution is converted into a bimodal distribution of respectively lighter and heavier "HDL"-particles than the normal ones; the densities at the peaks of these distributions are approximately 1.07 g/ml (light), 1.20 g/ml (heavy) and 1.11 g/ml (normal HDL). The optical extinction coefficient is apparently the same for the proteins of normal--and modified HDL. 6. Even high CRM concentrations (less than or equal to 116 mg/ml) have no perceptible effect on the gradient positions and profile of human serum albumin (HSA) and/or other heavy proteins. 7. The possible biological significance of these findings is briefly touched upon.     

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass

Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradients were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of LDL subclass pattern.     

Concentration and composition of serum lipoproteins in the fetal rat]

1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.     

Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions

Procedures are described for the isolation of lipoproteins from human serum by precipitation with polyanions and divalent cations. A mixture of low and very low density lipoproteins can be prepared without ultracentrifugation by precipitation with heparin and either MnCl(2) alone or MgCl(2) plus sucrose. In both cases the precipitation is reversible, selective, and complete. The highly concentrated isolated lipoproteins are free of other plasma proteins as judged by immunological and electrophoretic methods. The low density and very low density lipoproteins can then be separated from each other by ultracentrifugation. The advantage of the method is that large amounts of lipoproteins can be prepared with only a single preparative ultracentrifugation. Polyanions other than heparin may also be used; when the precipitation of the low and very low density lipoproteins is achieved with dextran sulfate and MnCl(2), or sodium phosphotungstate and MgCl(2), the high density lipoproteins can subsequently be precipitated by increasing the concentrations of the reagents. These lipoproteins, containing small amounts of protein contaminants, are further purified by ultracentrifugation at d 1.22. With a single preparative ultracentrifugation, immunologically pure high density lipoproteins can be isolated from large volumes of serum.     

Neutral lipid transfer and lipolysis convert high molecular weight LDL from cholesterol-fed nonhuman primates towards normal: a molecular analysis

In cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet, large, cholesterol ester-rich LDL (Mr greater than 3.5.10(6] are found at the same time that the plasma triacylglycerol levels are low. We studied whether the presence of higher concentrations of triacylglycerol-rich lipoproteins (VLDL) during in vitro incubations would allows depletion from LDL of cholesterol ester and a decreased LDL molecular weight. Three high Mr LDL (Mr = (3.7-4.8).10(6)), rich in cholesterol ester (50 +/- 1.4% by weight), were isolated from three animals by zonal ultracentrifugation, and were then incubated with human VLDL at 37 degrees C for 18 h in lipoprotein-deficient human plasma containing neutral lipid transfer activity. After incubation, modified LDL (M-LDL) was isolated by zonal ultracentrifugation. M-LDL was triacylglycerol-rich (36 +/- 5% by weight) and cholesterol ester-poor (20 +/- 3%), and cholesterol ester had transferred into VLDL. Purified lipoprotein lipase was added to the M-LDL, and triacylglycerol was hydrolyzed. The size of the post-lipolysis M-LDL (Mp-LDL) particles became smaller (mean diameters of 253 A and 228 A for two native LDLs and 215 A and 193 A for Mp-LDL, respectively). Both analytical and zonal ultracentrifugation showed Mp-LDL to be more dense than native LDL. Estimated molecular weights for Mp-LDL were 40%-50% less than that of the original LDL, and fell within the molecular weight range for normal human and monkey LDL. Lipid exchanges, but not apoprotein transfers, were responsible for LDL remodelling, as supported by three separate methods of analysis. Cholesterol ester losses accounted for about two-thirds of the molecular weight decrease. These in vitro results suggest that cholesterol ester enrichment of apoprotein B lipoprotein particles can be reversed by providing adequate levels of VLDL in the presence of neutral lipid transfer processes and lipolytic activity.     

Factors affecting the esterification of lipoprotein cholesterol by lecithin: cholesterol acyl transferase.

Lecithin: Cholesterol Acyltransferase (LCAT) esterified relatively small amounts of cholesterol from very low density lipoproteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). On the other hand, in the presence of very high density (>1.225 g/ml) plasma fraction (F-4), the enzyme esterified cholesterol from VLDL at considerably higher rates than from LDL or HDL. VLDL together with some component present in the very high density plasma fraction (F-4) may thus provide a highly efficient complex resulting in a favorable configuration of substrate lipids for the enzyme.