Role of LDL subfraction heterogeneity in the reduced binding of low density lipoproteins to arterial proteoglycans in cynomolgus monkeys fed a fish oil diet.

Previous studies using cynomolgus monkeys have shown that isocaloric substitution of dietary fish oil for lard reduced the in vitro binding of plasma low density lipoproteins (LDL) to arterial proteoglycans (PG) (Edwards, I.J., A.K. Gebre, W. D. Wagner, and J. S. Parks. 1991. Arterioscler. Thromb., 11: 1778-1785). The purpose of the present study was to determine whether all LDL subfractions were equally affected by the type of dietary fat with regard to PG binding and to identify compositional changes in LDL subfractions that might relate to the differential in PG binding. Two groups of cynomolgus monkeys (n = 5 each) were fed atherogenic diets (40% calories as fat; 0.26 mg cholesterol/kcal) containing 20% of calories as egg yolk and 20% as either lard or menhaden fish oil. LDL were isolated from plasma by ultracentrifugation and size exclusion chromatography and subfractionated by density gradient centrifugation. Three density ranges of LDL subfractions were collected from the gradients for determination of chemical composition, apoE and apoB content by ELISA, and binding to arterial PG in vitro. The d 1.015-1.025 g/ml subfraction contained 39 +/- 8% of the LDL cholesterol in the lard group but only 7 +/- 3% for the fish oil group. Values for cholesterol distribution were opposite for the d 1.035-1.045 g/ml subfraction, 8 +/- 1% versus 41 +/- 8%, respectively. Similar trends were noted for the distribution of apoB. For the lard group, LDL binding to arterial PG increased with decreasing density (i.e., increasing size) of the subfractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL (Sf greater than 9) and a decrease in the mass of denser LDL. LDL (1.019 less than d less than 1.063) were subdivided into two subfractions LDL1 (1.019 less than d less than 1.035) and LDL2 (1.035 less than d less than 1.063) by ultracentrifugation. The lipoproteins in these two fractions could be shown to have different flotation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL1 prior to its appearance in LDL2. [125I]LDL1 injected into the monkey was removed from the LDL1 density subclass with a half-life of 5.5-10.3 h. Much of the radioactivity injected as LDL1 was converted to denser LDL (LDL2). Labeled LDL2 injected into the monkey was not converted to LDL1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL2).     

Effects of rosuvastatin on electronegative LDL as characterized by capillary isotachophoresis: the ROSARY Study

Electronegative LDL, a charge-modified LDL (cm-LDL) subfraction that is more negatively charged than normal LDL, has been shown to be inflammatory. We previously showed that pravastatin and simvastatin reduced the electronegative LDL subfraction, fast-migrating LDL (fLDL), as analyzed by capillary isotachophoresis (cITP). The present study examined the effects of rosuvastatin on the more electronegative LDL subfraction, very-fast-migrating LDL (vfLDL), and small, dense charge-modified LDL (sd-cm-LDL) subfractions. Patients with hypercholesterolemia or those who were being treated with statins (n = 81) were treated with or switched to 2.5 mg/d rosuvastatin for 3 months. Rosuvastatin treatment effectively reduced cITP cm-LDL subfractions of LDL (vfLDL and fLDL) or sdLDL (sd-vfLDL and sd-fLDL), which were closely related to each other but were different from the normal subfraction of LDL [slow-migrating LDL (sLDL)] or sdLDL (sd-sLDL) in their relation to the levels of remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) C-II, and apoE. The percent changes in cm-LDL or sd-cm-LDL caused by rosuvastatin were correlated with those in the particle concentrations of LDL or sdLDL measured as LDL-apoB or sdLDL-apoB and the levels of HDL-C, RLP-C, apoC-II, and apoE. In conclusion, rosuvastatin effectively reduced both the vfLDL subfraction and sd-cm-LDL subfractions as analyzed by cITP.     

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.     

Study of the atherogenic dyslipoproteinemia induced by dietary cholesterol in rhesus monekys (Macaca mulatta).

Hypercholesterolemia was induced in adult male rhesus monkeys with a high-fat diet containing an elevated cholesterol level (0.5%). Plasma lipoproteins were chromatographically separated into four size populations (regions) that were subdivided by density until fractions with single electrophoretic mobilities were obtained. The region III lipoproteins (LDL) contained 80% of plasma cholesterol and were present in the highest concentration of all fractions. Their molecular weight was increased over that of controls so that each particle averaged 1.8 times the number of cholesteryl ester molecules as did control LDL. Region II lipoproteins, a heterogeneous group, were present in next highest concentration. Most were cholesteryl ester-rich, beta-migrating lipoproteins that overlapped the VLDL and LDL density ranges; apoB was the predominant apoprotein. One region II subfraction had pre beta 2 migration and the density range. 1.050 less than d less than 1.10. Another subfraction, cholesteryl ester-rich VLDL including only about 1% of plasma cholesterol, had pre beta 1 migration and apoB and apoC as the predominant apoproteins with no apoprotein E. Region I lipoproteins were larger sized, slow beta-migrating cholesteryl ester-rich VLDL that included 5% of plasma cholesterol. ApoB and apoE were the predominant apoproteins. Region IV lipoproteins (HDL) contained 4% of the plasma cholesterol; their concentration was decreased to about 1/3 of the control level. Atherogenic features of the diet-induced dyslipoproteinemia included the increased plasma concentrations and cholesteryl ester contents of the region I, II, and III lipoproteins in addition to the decreased HDL concentration.     

Metabolic behavior of hepatic VLDL and plasma LDL apoB-100 in African green monkeys

Recently, evidence has accumulated suggesting that significant amounts of plasma low density lipoproteins (LDL) may be derived by direct production. These plasma very low density lipoprotein (VLDL)-independent sources include the production and secretion of LDL-like particles directly by the liver, and/or a small pool of nascent precursor particles that are converted rapidly to LDL. The current studies were designed to test the hypothesis that hepatic VLDL represent a rapidly turning over precursor pool to plasma LDL in African green monkeys. Livers from African green monkeys were perfused with serum-free medium containing [3H]leucine or 3H-labeled amino acids for 4-6 hr. Hepatic [3H]VLDL and autologous plasma 125I-labeled LDL were injected simultaneously into recipient animals and density gradient ultracentrifugation and gel filtration were used to characterize the distribution of 3H and 125I radioactivity at selected times after injection. These studies show that 4 to 66% of the injected dose of hepatic VLDL [3H]apoB-100 was metabolized extremely rapidly into particles that resembled the recipient's plasma LDL by size and density. Based on the kinetic model developed to describe the metabolic behavior of hepatic VLDL [3H]apoB-100, the estimated maximal pool size of hepatic VLDL apoB-100 in these animals was very small (0.042 and 0.112 mg) and represented, at best, approximately 10% of the average plasma VLDL apoB-100 mass found in cholesterol-fed African green monkeys. In addition, the radiolabeled hepatic LDL appear to be metabolized similarly to plasma LDL. That is, the rapid conversion of hepatic VLDL as well as the direct production of hepatic particles within the LDL density range appear to contribute to plasma LDL. Metabolic heterogeneity was also seen within the LDL class. The more buoyant subfraction (LDL1) had a higher turnover rate than the more dense subfraction (LDL2) and hepatic VLDL-derived [3H]LDL1 had a slower final rate of plasma disappearance than the plasma-derived 125I-labeled LDL1 in most animals. The results from these studies suggest that a small pool of hepatic VLDL can be converted very rapidly to plasma LDL and may contribute significantly to the large plasma pool of LDL seen in cholesterol-fed African green monkeys. This pathway may be analogous to the pathway in some human subjects in which a portion of human plasma VLDL is converted rapidly into LDL without passing through a delipidation cascade, often referred to as direct LDL production.     

Rapid isolation of VLDL subfractions: assessment of composition and susceptibility to copper-mediated oxidation

VLDLs, synthesized and released by the liver, are a heterogeneous group of particles of varying composition and metabolic fates. A method is described for the rapid isolation of VLDL into four subfractions (A-D) and assessment of their susceptibility to oxidation. The total isolation procedure required less than 3.5 h, and was achieved by gradient ultracentrifugation. Each subfraction was assessed for triglyceride, cholesterol, and apolipoprotein B (apoB) composition and for the presence of contaminants such as albumin and urate. The oxidation potential, in the presence of copper ions, of each subfraction was also assessed. This rapid procedure produced VLDL fractions analogous to those produced by a previously reported but more prolonged isolation method. Comparison of the two procedures demonstrated that lipid and apoB were similar, while the rapid procedure produced subfractions void of albumin and urate contamination and lower in preformed hydroperoxides. Compositional changes were found between the subfractions: as the subfractions became smaller and more dense (A-->D), there was a decrease in the ratio of triglyceride to apoB and an increase in the ratio of cholesterol to apoB, also arachidonic acid was increased in subfraction D compared with subfractions A, B, and C. The smaller subfractions were more susceptible to oxidation, a trend similar to that reported previously for the oxidation of LDL subfractions.     

Single spin density gradient ultracentrifugation method for the detection and isolation of light and heavy low density lipoprotein subfractions

A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.     

High density lipoprotein accumulation in perfusates of isolated livers of African green monkeys. Effects of saturated versus polyunsaturated dietary fat

To determine whether altered hepatic secretion of HDL is part of the mechanism by which polyunsaturated fat lowers plasma HDL concentration, we have studied HDL secretion in the isolated perfused livers of African green monkeys fed an atherogenic diet containing either safflower oil as the polyunsaturated fat or butter as the saturated fat. During recirculating perfusion with a lipoprotein-free medium, livers from safflower oil-fed animals produced 21% less HDL mass on the average than those from butter-fed animals. Newly secreted hepatic HDL were characterized after their isolation and subfractionation by a combination of agarose column chromatography and density gradient ultracentrifugation. In both diet groups the HDL were heterogeneous in size, morphology, and composition and consisted of discoidal particles ranging in diameter from greater than 200 A to as little as 50 A. Large, discoidal particles that were rich in apoE and apoA-I were separated from small particles that were poor in apoE but rich in apoA-I. All hepatic HDL subfractions contained only small amounts of cholesteryl ester and triglyceride. The hepatic particles resembled in composition and structure the large variety of HDL particles found in the plasma of patients with the familial deficiency of lecithin:cholesterol acyltransferase. Accordingly, perfusate LCAT activity was measured and found to be 2% or less than that in monkey plasma. We conclude that the perfused monkey liver produces a variety of nascent HDL that are relatively unmodified by the post-secretory metabolic events which normally occur in blood plasma in vivo, and that livers of polyunsaturated fat-fed monkeys secrete fewer plasma HDL precursor particles than do those of saturated fat-fed monkeys.     

Alterations of high density lipoproteins in experimental intrahepatic cholestasis in the rat induced by administration of alpha-naphthylisothiocyanate

It has previously been reported that abnormally enlarged high density lipoproteins (HDL) appear in rats with extrahepatic cholestasis induced by ligation of the common bile duct. To see whether similar changes in HDL occur in intrahepatic cholestasis in rats, we studied HDL alterations in rats treated with alpha-naphthylisothiocyanate (ANIT), which is known to produce a cholestatic response in rats similar to intrahepatic cholestasis in man. Findings were obtained which indicated changes in HDL similar to those in bile duct-ligated rat serum: HDL from ANIT-treated rats were separated into two subfractions, enlarged particles and smaller ones, on Bio-Gel A5m column chromatography. In electron micrographs, the two subfractions appeared spherical and the diameters of the enlarged particles and the other ones were 15.0 +/- 2.6 nm and 11.5 +/- 2.2 nm, respectively. Both subfractions showed slow alpha-mobility in agarose gel electrophoresis. The enlarged HDL had apoE as their major apoprotein, while apoA-I was the major apoprotein in the other HDL subfraction. The enlarged HDL contained less protein and more cholesterol than the other HDL subfraction. The two HDL subfractions were also separated by heparin-Sepharose affinity chromatography.     

The fatty acid distribution in low density lipoprotein in diabetes.

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

Changes in plasma very low density and low density lipoprotein content, composition, and size after a fatty meal in normo- and hypertriglyceridemic man.

Four subfractions of plasma VLDL characterized by decreasing Sf value and LDL were isolated by density gradient preparative ultracentrifugation from normotriglyceridemic (NTG) and hypertriglyceridemic (HTG) (type IV) subjects in the fasting state and after a fatty meal. Chemical analysis and computation of numbers of particles in each fraction showed that the hyperlipidemia of type IV subjects was accounted for by an increase in total numbers of VLDL and a shift in the distribution of VLDL towards particles of larger diameter. Postprandial hyperlipidemia was due to the presence of chylomicron remnants rather than intact chylomicrons, and was accounted for by an increase in particle diameter of the largest VLDL subfraction rather than by an increase in particle numbers. Postprandial hyperlipedemia was accompanied by a shift in the distribution of VLDL towards particles of larger diameter in both NTG and HTG subjects, probably because of competition for the triglyceride-depletion process between chylomicrons and hepatic VLDL. Most chylomicron remnants were removed from the circulation without degradation to smaller VLDL or to LDL, but some remnants were sufficienty small to contribute to smaller VLDL subfractions. The LDL of type IV subjects contained more apoprotein B than those from NTG subjects, and this difference was associated with increases in diameter, molecular weight, density, and the ratio of protein: phospholipid in LDL from type IV subjects. Defective degradation of large VLDL to small VLDL, and of VLDL to LDL may be related to this alteration in apoprotein B content of the lipoproteins in type IV subjects.     

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.     

The fatty acid distribution in low density lipoprotein in diabetes

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the CETP inhibitor anacetrapib

We investigated the effects of the cholesteryl ester (CE) transfer protein inhibitor anacetrapib (ANA) on plasma lipids, lipoprotein subfraction concentrations, and lipoprotein composition in 30 healthy individuals. Participants (n = 30) were randomized to ANA 20 mg/day, 150 mg/day, or placebo for 2 weeks. Changes in concentration of lipoprotein subfractions were assessed using ion mobility, and compositional analyses were performed on fractions separated by density gradient ultracentrifugation. ANA 150 mg/day versus placebo resulted in significant decreases in LDL-cholesterol (26%) and apo B (29%) and increases in HDL-cholesterol (82%). Concentrations of medium and small VLDL, large intermediate density lipoprotein (IDL), and medium and small LDL (LDL2a, 2b, and 3a) decreased whereas levels of very small and dense LDL4b were increased. There was enrichment of triglycerides and reduction of CE in VLDL, IDL, and the densest LDL fraction. Levels of large buoyant HDL particles were substantially increased, and there was enrichment of CE, apo AI, and apoCIII, but not apoAII or apoE, in the mid-HDL density range. Changes in lipoprotein subfraction concentrations and composition with ANA 20 mg/day were similar to those for ANA 150 mg/day but were generally smaller in magnitude. The impact of these changes on cardiovascular risk remains to be determined.     

Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation

The molecular basis of the heterogeneity of plasma low density lipoproteins (LDL, d 1.024-1.050 g/ml) was evaluated in 40 normolipidemic male subjects following fractionation by isopycnic density gradient ultracentrifugation into eight major subspecies. The mass profile of our subjects' LDL uniformly displayed single symmetric or asymmetric peaks as a function of density; the peak occurred most frequently (20 subjects) in subfraction 7 (d 1.0297-1.0327 g/ml). Several physicochemical properties (hydrodynamic behavior, electrophoretic mobility, chemical composition, size and particle heterogeneity, and apolipoprotein heterogeneity) of the LDL subfractions were examined. Hydrodynamic analyses revealed unimodal distributions and distinct peak Sf degree rates in individual subfractions. Such behavior correlated well with particle size and heterogeneity data, in which LDL subspecies were typically resolved as unique narrow bands by gradient gel electrophoresis. Subspecies with average densities of 1.024 to 1.0409 g/ml ranged from 229 to 214 A in particle diameter. LDL protein content increased in parallel with density while the proportion of triglyceride diminished; cholesteryl esters predominated, accounting for approximately 40% or more by weight. Distinct differences in net electric charge were demonstrated by electrophoresis in agarose gel, the subspecies with average density of 1.0314 g/ml displaying the lowest net negative charge. ApoB-100 was the major apoprotein in all subspecies, and constituted the unique protein component over the density interval 1.0271-1.0393 g/ml. ApoE and apo[a] were detected at densities less than 1.0271 and greater than 1.0393 g/ml. While apoE was evenly distributed within these two regions, representing up to 2% of apoLDL, the distribution of apo[a] was skewed towards the denser region, in which it amounted to 3-7% of apoLDL. ApoC-III was detectable as a trace component at densities greater than 1.0358 g/ml. Calculation of the number of molecules of each chemical component per LDL subspecies showed the presence of one copy of apoB-100 per particle, in association with decreasing amounts of cholesteryl ester, free cholesterol, and phospholipid. These data indicate that a similar overall molecular organization and structure is maintained in a unimodal distribution of LDL particle subspecies over the density range approximately 1.02 to 1.05 g/ml. In sum, our data may be interpreted to suggest that microheterogeneity in the physicochemical properties of human LDL subspecies reflects dissimilarities in their origins, intravascular metabolism, tissular fate, and possibly in their atherogenicity.     

Guinea pig low density lipoproteins: structural and metabolic heterogeneity

The structural and metabolic heterogeneity of low density lipoproteins (LDL, d 1.024-1.100 g/ml) has been investigated in the guinea pig. Two LDL subfractions, of d 1.024-1.050 and 1.050-1.100 g/ml, respectively, were isolated by sequential ultracentrifugation; while both were enriched in cholesteryl ester and apoB-100, the former was heterogeneous displaying three particle size species of diameters 26.9, 25.6, and 24.7 nm, whereas the denser subfraction was relatively homogeneous containing a single, smaller species (diam. 23.6 nm). The fractional catabolic rates (FCR) of the two LDL subfractions were alike (approximately 0.090 pools/hr) in the guinea pig in vivo. After modification of each subfraction by reductive methylation, the FCRs were reduced similarly and indicated that 70-80% of degradation occurred via the cellular LDL receptor pathway. However, the intravascular metabolism of these LDL subfractions, determined from the radioactive content of density gradient fractions as a function of time after injection of radiolabeled native or chemically modified LDL, tended to be distinct. Thus, while radiolabeled apoB-100 in the lighter subfraction maintained the initial density profile up to 48 hr, the radioactive profile of its methylated counterpart changed, the proportion of radioactivity in the lighter gradient fractions (d 1.027-1.032 g/ml) increasing while that in the denser (d 1.037-1.042 g/ml) fractions diminished. A more marked transformation occurred in LDL of d 1.050-1.100 g/ml, in which the radioactive profile shifted towards lighter particles of the d 1.024-1.050 g/ml species; this shift was partially dependent on the LDL receptor, since it was more pronounced in the methylated subfraction. Furthermore, a net increase in the radioactive content of gradient subfractions 7 to 9 (d 1.032-1.042 g/ml) was found 10 hr after injection of methylated LDL of d 1.050-1.100 g/ml, at which time the bulk of LDL radioactivity had been removed from plasma. Several mechanisms, acting alone or in combination, may account for these findings; among them, some degree of transformation of dense to lighter LDL species appears a prerequisite. In conclusion, our data attest to the structural heterogeneity of circulating LDL in the guinea pig, and suggest that the intravascular processing and metabolism of LDL particle subspecies is directly related to their structure and physicochemical properties.