Low Density Lipoprotein Subspecies Research Articles

Conformational differences in human apolipoprotein B-100 among subspecies of low density lipoproteins (LDL). Association of altered proteolytic accessibility with decreased receptor binding of LDL subspecies from hypertriglyceridemic subjects.

We asked at what point in the metabolic cascade of very low density lipoproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the effect of hypertriglyceridemia on the proteolytic accessibility, secondary structure, and receptor-binding affinity of B-100 in LDL subspecies of varying density. Limited proteolysis with Staphylococcus aureus V8 protease and cathepsin D identified the density (about 1.033 g/ml) between two LDL subspecies, designated LDL-1 and -2, as the transition point during VLDL metabolism of both normolipidemic (N-) and hypertriglyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia greatly altered proteolytic accessibility of B-100 in the denser LDL subspecies. Specifically, B-100 in HTG-LDL exposed more cleavage sites than in N-LDL, including two novel sites, approximately 120 and approximately 130 kDa from the NH2 terminus in the small and dense subspecies (designated LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dichroic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HTG-LDL. Binding affinity for the LDL receptor of human fibroblasts decreased markedly with increasing density among HTG-LDL subspecies (by approximately 50% for LDL-4.5 or -5). We conclude that the changes in proteolytic accessibility observed between LDL-1 and -2 and in LDL-4, -4.5, or -5 indicate significant differences in local conformation of B-100 at specific peptide regions. The association of exposure of more cleavage sites, especially novel sites in the NH2-terminal regions, with greatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests that altered local conformation in B-100 apart from the putative receptor-binding domain might affect interaction with the receptor.

Preferential cholesteryl ester acceptors among the LDL subspecies of subjects with familial hypercholesterolemia.

Elevated cholesteryl ester transfer protein-mediated transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) may contribute to the atherogenicity of LDL in subjects with familial hypercholesterolemia (FH). To identify the major CE acceptors among LDL subspecies, we investigated the qualitative and quantitative features of CE transfer and exchange to LDL on incubation of plasma under physiological conditions. LDL subspecies were fractionated by density-gradient ultra-centrifugation. Both mass transfer and exchange of HDL CE to and with very-low-density lipoprotein plus intermediate-density lipoprotein and LDL were linear for the first 6 hours of incubation. Thereafter mass transfer ceased, but exchange continued at a comparable rate. The rate of CE mass transfer to apolipoprotein B-containing lipoproteins was significantly enhanced in heterozygous FH subjects compared with normolipidemic individuals (91.6 +/- 28.2 versus 52.9 +/- 19.6 micrograms CE/h per milliliter plasma, FH versus normal subjects, P < .02). In FH subjects the predominant LDL subspecies (LDL 3 and 4, d = 1.029 to 1.050 g/mL) accounted for 59.7 +/- 9.2% of the total CE transferred to LDL from HDL. By contrast, expression of CE mass transfer relative to the mass of each lipoprotein acceptor showed the triglyceride (TG)-rich (10.7% to 17.3%), light LDL subspecies (LDL 1 and 2, d = 1.019 to 1.029 g/mL) to represent the preferential CE acceptors (LDL 1 and 2, 94.8 to 136.5 micrograms CE/mg LDL mass; LDL 3 through 5 [d = 1.029 to 1.063 g/mL], 47.1 to 64.1 micrograms CE/mg LDL mass).(ABSTRACT TRUNCATED AT 250 WORDS)

Ciprofibrate therapy normalises the atherogenic low-density lipoprotein subspecies profile in combined hyperlipidemia

The effect of ciprofibrate treatment on the atherogenic profile of low-density lipoprotein (LDL) subspecies in combined hyperlipidemia (CHL) has been investigated in six patients displaying elevated plasma triglyceride and cholesterol levels ( > 200 and > 250 mg/dl, respectively). The E 2E 2 phenotype was excluded; four patients possessed familial antecedents of premature coronary heart disease (CHD). Analysis of five LDL subclasses separated by isopycnic density gradient ultracentrifugation showed a predominance of dense LDL subspecies (LDL-4 and LDL-5, d 1.039–1.063 g/ml; 51% of total LDL mass) in the asymmetric LDL density profile characteristic of CHL patients at baseline. Ciprofibrate treatment (100 mg/day for 1 month) effected marked reductions in both total plasma LDL and apo B-100 levels ( ∼ 19% and ∼ 23%, respectively). Equally, the plasma profile of LDL subspecies was normalised to a significant degree as a result of preferential reduction in the elevated levels of both dense subspecies (LDL-4 and LDL-5; −43% and −54%, respectively; P < 0.03 and P < 0.06, respectively). The circulating concentrations of light LDL (LDL-1, d 1.019-1.023 g/ml) were also diminished significantly by ciprofibrate (−30%; P < 0.06). Furthermore, ciprofibrate not only effected reductions in the elevated triglyceride content of the hydrophobic core of all LDL subspecies but also normalised their common deficiency in free cholesterol. In addition, the abnormally small particle diameters of LDL-4 and −5 were increased to normal. Plasma levels of both apo B-100 and triglycerides were significantly and positively correlated with those of LDL-4 and LDL-5, suggesting not only that the degree of triglyceride elevation is intimately linked to the extent of shift in LDL subclass profile towards denser subspecies, but also that triglyceride reduction upon treatment strongly influences LDL-4 and LDL-5. In conclusion, our findings indicate that ciprofibrate treatment in combined hyperlipidemia results in marked reduction in plasma triglyceride levels (-33%), and that such reduction is intimately linked to normalisation of both the qualitative and quantitative features of the atherogenic LDL subspecies profile typical of this disorder.

Characteristics of low-density lipoprotein subfractions from patients with coronary artery disease.

The low-density lipoprotein (LDL) of patients with coronary artery disease (CAD) has been reported to enhance cholesterol ester accumulation in aortic cells. The LDL of patients with CAD also has a higher mean density than the LDL of healthy subjects, indicating a different subspecies distribution. Because these density differences may be associated with altered metabolism and atherogenicity of LDL, we studied the properties and effects of isolated LDL subfractions on cell lipid metabolism and cholesterol accumulation. Plasma pools of patients with angiographically proven CAD (A) and healthy controls (C) were used to isolate and fractionate LDL into five subfractions (1 to 5, from the lowest to the highest density) by gradient ultracentrifugation. Each of the LDL subfractions was analyzed for particle diameter, chemical composition, sialic acid content, and their effect on lipid content and cholesterol esterification in arterial cell and macrophage cultures, respectively. The chemical compositions of the respective subfractions revealed no differences between patients and controls, except that the sialic acid content was reduced in dense LDL subfractions, especially in the samples from patients with CAD (fractions A3, A4, A5, and C5). LDL subfractions with reduced sialic acid content also enhanced the incorporation of [14C]-oleate into cholesterol esters in mouse peritoneal macrophage cultures (fractions A4, A5, and C5) and increased the cholesterol ester content in primary cultures of human aortic intimal cells (fractions A3, A4, A5, and C5). The results suggest that the dense LDL subfractions of patients with CAD are atherogenic by promoting intracellular cholesterol ester accumulation. The results also suggest that the atherogenicity is associated with reduced sialic acid content of the LDL subspecies.

Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia.

Patients presenting combined hyperlipidemia (CHL) display an elevated risk of coronary heart disease. The atherogenic lipoprotein particles implicated in this disorder remain ill defined. We determined the qualitative and quantitative characteristics of the density distribution of low density lipoprotein (LDL) particle subspecies in nine subjects defined phenotypically as presenting CHL, and under strict dietary control. Seven CHL patients possessed familial antecedents of premature coronary heart disease; none were E2E2 homozygotes. Five LDL subspecies were isolated by density gradient ultracentrifugation in the density range 1.019-1.063 g/ml. In all patients, the LDL profile was skewed towards the dense subspecies (LDL-4, d 1.039-1.050 g/ml and LDL-5, d 1.050-1.063 g/ml), representing 47% of total LDL mass; by contrast, these subspecies accounted for only 30% of LDL mass in five normolipidemic subjects (P < 0.01). In addition, plasma LDL mass concentrations were some twofold higher in CHL patients as compared to normolipidemic subjects. The % mass of LDL-4 was positively correlated with plasma triglyceride and apoB levels. LDL-2 and LDL-3 in CHL patients were triglyceride-enriched (11.9 and 7.2%, respectively) as compared to the corresponding subspecies in normolipidemic subjects (6.6 and 3.7%, respectively; P < 0.05 in each case). LDL particle size decreased with increase in density in both groups; however, significant differences were found between corresponding LDL subspecies (LDL-1, -3, -4, and -5) in CHL patients and normolipidemic subjects, a finding suggestive of dissimilar molecular organization, despite correspondence in hydrated density. The copper-induced oxidative modification of LDL subspecies was assessed by determination of conjugated diene formation. In both groups, LDL-5 was distinct in exhibiting a marked diminution in oxidative resistance as indicated by a significant reduction (P < 0.01) in mean lag time. The oxidative susceptibility of LDL subspecies in both groups was independent of vitamin E content when expressed as the ratio vitamin E/LDL mass, although dense LDL in CHL patients tended to be deficient in this antioxidant. The diminished oxidative resistance of dense LDL subspecies could not be accounted for by enrichment in polyunsaturated fatty acids in either group. These studies suggest that in consequence of their elevated circulating concentration and diminished oxidative resistance, dense LDL subspecies represent putative atherogenic subspecies in combined hyperlipidemia.

Alcohol dose and low density lipoprotein heterogeneity in squirrel monkeys ( Saimiri sciureus)

The present study was designed to determine whether normolipidemic male squirrel monkeys ( Saimiri sciureus) exhibit low density lipoprotein (LDL) heterogeneity similar to that observed in humans and if present, whether LDL subfractions are altered by consumption of low vs. high dose ethanol (EtOH). Primates were divided into three groups designated control, low, and high EtOH and fed isocaloric liquid diets containing 0%, 12% and 24% of calories as EtOH, respectively, for 6 months. The 12% EtOH caloric level resulted in a modest, non-significant increase in high density lipoprotein (HDL) cholesterol and no change in LDL cholesterol or plasma apolipoprotein B (apo B), while the 24% dose produced significant elevations in plasma, LDL and HDL cholesterol and apo B. Using a single-spin density gradient ultracentrifugation procedure developed for humans, three distinct LDL subclasses designated LDL 1a ( d = 1.031 g/ml), LDL 1b ( d = 1.038 g/ml) and LDL 2 ( d = 1.046 g/ml) were isolated from all three treatment groups. Monkey LDL subfractions were nearly identical to very light, light and heavy LDL subspecies isolated from human plasma in terms of their: (1) isopycnic densities following ultracentrifugation; (2) co-migration as single bands with beta-electrophoretic mobility in cellulose acetate and agarose electrophoretic gels; (3) size-dependent migration pattern in polyacrylamide gradient electrophoretic gels; (4) co-migration as a single band corresponding to apo B-100, following SDS polyacrylamide gel electrophoresis; and (5) decrease in total cholesterol/protein ratios with increasing LDL subclass density. Although there were no treatment differences in LDL particle size, within each treatment group, mean particle size for each LDL subfraction was significantly different from every other subfraction. Low (12%) dose alcohol had no effect on LDL subfraction mass relative to controls while high alcohol consumption resulted in marked increases in all lipid (except triglyceride) and protein of the larger, buoyant LDL subspecies (LDL 1a and LDL 1b). Moreover, the best correlation between plasma apo B and LDL subfraction total mass was demonstrated with LDL 1b ( r = 0.735). Since neither the lipid nor the protein concentration of the small, dense, purportedly more atherogenic, LDL 2 changed with the 24% EtOH dose, we propose that the LDL subfraction alterations associated with high alcohol intake in squirrel monkeys (increased LDL 1a, increased LDL 1b, LDL 2 no effect) may represent a compensatory response to modulate the overall atherogenic lipoprotein profile associated with elevations in total LDL cholesterol and plasma apolipoprotein B.

Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.

The low density lipoproteins (LDL) of human plasma consist of a series of discrete particle subspecies of distinct physicochemical, immunological, and hydrodynamic properties. Such structural differences are intimately linked to the metabolic heterogeneity of circulating LDL in vivo. The current studies were designed to evaluate and compare the interaction of discrete LDL subspecies from normolipidemic subjects with the LDL receptor. Plasma LDL of d 1.019-1.063 g/ml from healthy males were fractionated into 15 subspecies of defined physicochemical characteristics by isopycnic density gradient ultracentrifugation as described earlier (Chapman et al., J. Lipid Res. 1988. 29: 442-458). The major LDL subspecies, LDL-5 to LDL-10, exhibited an overall range in density from 1.0244 to 1.0435 g/ml; individual subspecies increased in density by increments of 0.027 (LDL-5), 0.026 (LDL-6), 0.030 (LDL-7), 0.031 (LDL-8), 0.035 (LDL-9), and 0.042 g/ml (LDL-10), respectively. Taken together, these subspecies accounted for approximately 70% of the total mass of LDL of d 1.019-1.063 g/ml; their cholesterol: protein ratios decreased from 1.70 to 1.12 and particle size from 275 to 260 A with increase in density. ApoB-100 was the unique protein component in subspecies 5-8, with trace amounts (less than 0.2% of apoLDL) of both apoA-I and apoE in subspecies 9 and 10. The interaction of individual LDL subspecies with the LDL receptor on cultured human U-937 monocyte-like cells was compared by determining receptor binding affinities at 4 degrees C. Scatchard analysis of specific binding curves demonstrated a single class of binding site for each subspecies. The lowest dissociation constants were displayed by LDL subspecies 6 (Kd 5.71 nM), 7 (Kd 5.24 nM) and 8 (Kd 4.67 nM), while subspecies 5, 9, and 10 displayed significantly higher Kd values (8.35, 7.20, and 6.87 nM, respectively). Competitive displacement studies at 4 degrees C, in which unlabeled subspecies from the same gradient series competed for binding with 125I-labeled LDL subspecies, confirmed the relative binding affinities of these subspecies. As the hydrophobic lipid core of LDL undergoes a thermotropic transition at approximately 37 degrees C, which may in turn influence the surface structure of the particle, internalization and degradation studies were performed at 37 degrees C. No effect of temperature was detectable; again, LDL subspecies at each extreme of the density distribution (LDL-5 and LDL-10) displayed significantly lower binding affinities for the LDL receptor than that from the peak region (LDL-7).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of fluvastatin (XU 62-320), an HMG-CoA reductase inhibitor, on the distribution and composition of low density lipoprotein subspecies in humans

We studied the effect of fluvastatin (XU 62-320), a new HMG-CoA reductase inhibitor, on the distribution of low density lipoprotein (LDL) subspecies and composition in humans. As expected, fluvastatin significantly lowered serum LDL levels (25% after 6 weeks of therapy). In addition, treatment with fluvastatin changed the LDL subspecies. In the group treated with fluvastatin, 38.5% of the individuals showed changes in the shape of LDL absorbance profile obtained from density gradient ultracentrifugation and 54% of the group showed changes in the electrophoretic mobility of the LDL bands. Of those showing changes in electrophoretic mobility, the majority (78%) shifted to slightly larger, less dense LDL after drug therapy. However, the LDL-cholesterol/apo B ratio changes were relatively small in all fluvastatin-treated individuals including the group with changes in eletrophoretic mobility, confirming that HMG-CoA reductase inhibitor causes relatively small and subtle changes in the distribution of LDL subspecies.

Overproduction of a buoyant low density lipoprotein subspecies in spontaneously hypercholesterolemic mutant pigs.

We previously described the hypercholesterolemia of pigs with defined apolipoprotein B (apo B) alleles associated with reduced binding of low density lipoprotein (LDL) to its receptor in vitro and slow clearance from the circulation in vivo. The increased plasma LDL in the hypercholesterolemic pigs was confined to a buoyant LDL subspecies. Because of this qualitative change in the LDL subspecies profile, we studied the turnover of buoyant and dense LDL subspecies independently. Normal and mutant radioiodinated buoyant and dense LDLs were simultaneously injected into normal and mutant pigs, and the clearance rates, interconversion rates, and production rates were determined. The sevenfold increase in buoyant LDL levels in the mutant pigs was due to a fivefold increase in buoyant LDL production. Total mutant LDL production was increased approximately 25%, suggesting that part of the increase in buoyant LDL production is at the expense of dense LDL production. Conversion of dense LDL to buoyant LDL made a small contribution to the buoyant LDL increase. The turnover analysis showed that dense LDL, in both mutant and control pigs, is primarily derived from a source other than buoyant LDL. To test this more directly, [3H]leucine was intravenously injected, and the specific activity of the LDL subspecies was measured over 96 hours. There was a large discrepancy in the areas under the specific activity-versus-time curves, indicating that buoyant LDL cannot be the sole precursor of dense LDL and further supporting the conclusion that buoyant and dense LDL are, in part, metabolically independent particles. These results show that genetic variation in the apo B locus can affect the synthetic rate of LDL and the LDL subspecies distribution.

Increased immunoreactivity of apolipoprotein B epitopes during prolonged storage of low density lipoproteins

Human low density lipoproteins (LDL) serve as the major carriers of plasma cholesterol. LDL are heterogeneous in their lipid content, size and density [l] and certain LDL subspecies may increase risk of atherosclerosis [l], possibly due to differences in the conformation of apolipoprotein (APO) B in the particle. Attempts to determine the structural organization of Apo B in LDL have been largely frustrated due to the extreme insolubility and aggregation of delipidated apo B. More recent investigations have utilized monoclonal antibodies (MAbs) [2,3] to probe apo B structure in LDL. A number of these investigations have established that apo B is immunochemically heterogeneous [2,3] and that alterations in the lipid [2] or protein [3] components of LDL result in changes in immunoreactivity. In the present study we report our finding that the LDL isolated from some, but not all, individuals show a time related increase in enzyme-linked immunosorbent assay (ELISA) rates for three monoclonal antibodies.

Rapid isolation of low density lipoprotein (LDL) subfractions from plasma by density gradient ultracentrifugation

High resolution density gradient ultracentrifugation (DGUC) and non-denaturing gradient gel electrophoresis (GGE) indicate that low density lipoprotein (LDL) in both normal and hyperlipidaemic subjects is composed of overlapping particle populations. A new centrifugation procedure has been developed which permits the separation of LDL subspecies directly from plasma within 24 h. The profiles obtained were analogous to those seen on gradient gel electrophoresis. LDL was divided into 3 fractions. The plasma concentration of LDL-I seen in young females was twice that in men (85.6 ± 28.8 vs. 42.3 ± 25.7 mg/dl, P < 0.005). LDL-II was not significantly different in any group while LDL-III was specifically elevated in coronary artery disease (CAD) patients (207.1 ± 92.6 mg/dl inCAD vs. 87.4 ± 79.6 mg/dl in normal men, P < 0.05). The presence of small, dense LDL detected either by density gradient centrifugation or gel electrophoresis was associated with raised triglyceride (TG) and low high density lipoprotein (HDL) cholesterol and may be a risk marker for coronary artery disease.

Defective receptor binding of low density lipoprotein from pigs possessing mutant apolipoprotein B alleles.

We previously identified a defect in the in vivo catabolism of low density lipoprotein (LDL) from hypercholesterolemic pigs carrying a mutant apolipoprotein B allele. In the present studies, we examined the in vitro metabolism of mutant LDL in cultured pig fibroblasts. A 3-fold higher concentration of mutant LDL (compared to control) was needed to displace 50% of control 125I-LDL binding. Mutant LDL had a 6-fold higher dissociation constant than control LDL. Scatchard plots of the binding data were concave upward, suggesting multiple classes of binding sites or negative cooperativity. The mutant LDL degradation rate was reduced by 40%; this decrease could be attributed to a dense LDL subspecies. Mutant and control buoyant LDL subspecies were degraded more slowly than the corresponding dense LDL subspecies. Together, these studies show that diminished LDL receptor binding can result from mutations in apolipoprotein B and from changes in the lipid composition of LDL particles.

Defective catabolism and abnormal composition of low-density lipoproteins from mutant pigs with hypercholesterolemia.

Metabolic and chemical properties of low-density lipoproteins (LDLs) were studied in a strain of pigs carrying a specific apo-B allele associated with hypercholesterolemia and premature atherosclerosis. LDL mass was significantly greater in mutant than in control pigs (400 +/- 55 mg/dL vs 103 +/- 26 mg/dL), as was LDL cholesterol. When normal and mutant LDLs were injected into the bloodstream of normal pigs, the fractional catabolic rate (FCR) of mutant LDL was about 30% lower than that of control LDL. In mutant pigs, the mean FCRs of mutant and control LDL were similar, although they were much lower than the corresponding FCRs observed in normal pigs. The density profile of LDL particles differed in control and mutant pigs; the peak LDL flotation rate was shifted from S0f = 5.3 +/- 1.9 in controls to a more buoyant 7.4 +/- 0.5 in mutants. The elevation of LDL in the mutants was restricted to the most buoyant LDL subspecies. This subpopulation of mutant LDL was enriched with cholesteryl ester (47% vs 37%) and depleted of triglyceride, relative to LDL of similar density and size in controls. The lipid compositions of the denser LDL subpopulations (rho greater than 1.043 g/mL) were similar in mutants and controls. We conclude that the hypercholesterolemia of these mutant pigs is accounted for by defective catabolism of LDL. The buoyant cholesterol ester enriched LDL subspecies that accumulate in plasma may contribute to the accelerated atherogenesis that occurs in these animals.

Low density lipoprotein metabolism in familial combined hyperlipidemia and familial hypercholesterolemia: Kinetic analysis using an integrated model

Several models for low density lipoprotein (LDL) apo B metabolism were applied to LDL turnover data from subjects with two distinct genetic forms of hyperlipidemia, familial hypercholesterolemia (FH), and familial combined hyperlipidemia (FCHL). Of the first two models tested, there was good agreement between the observed and predicted data for FH in one (model A), and for FCHL in the other (model B). The major difference between these two models is that LDL is kinetically homogenous in model A and heterogeneous in model B, raising the possibility that LDL subspecies differences may occur between these two disorders. The findings are consistent with LDL homogeneity in FH and LDL heterogeneity in FCHL. Two other integrated models (models C and D) provided good agreement between observed and predicted data in both disorders. Although neither could be rejected outright on the basis of known physiology, parameter estimates were more variable with model D. Analysis of the data using model C was consistent with the known pathophysiologic defect in LDL catabolism in FH and suggests that individuals with FH as well as FCHL have more than one LDL subpopulation in plasma. The urine/plasma ( U P ) ratio was shown to be constant from day 4 to day 14 of the study in FH, while in FCHL this value declined in all cases. Thus, determination of LDL fractional catabolic rates (FCR) by the U P ratio method may be invalid in certain groups of patients. The other traditional method for calculating LDL FCR, the Matthews' analysis, overestimated FCR in some instances, and could lead to systematic errors when used to determine LDL FCR and production rates. These studies point out some of the complexities of LDL metabolism that may relate to heterogeneity of LDL and their subspecies, and underscore the difficulty of accurately measuring LDL turnover parameters using either the U P ratio method or a simple model in which only plasma LDL decay curves are analyzed. These studies also point out the need to use an integrated model for LDL metabolism that utilizes both plasma and urine radioactivity data.

Presence of multiple subpopulations of lipoproteins of intermediate density in normal subjects.

Analysis of human plasma by gradient gel electrophoresis (GGE) revealed multiple distinct bands in the size range between very low density (VLDL) and low density lipoproteins (LDL). GGE of intermediate density lipoproteins (IDL) from normal subjects consistently showed two major bands with particle diameters in the range of 275 to 300 A. The larger, usually predominant subspecies was designated IDL-1, and the smaller, IDL-2. GGE also demonstrated two sizes of subpopulations in the VLDL density range in all subjects. A discontinuous nonequilibrium density gradient ultracentrifugation technique was devised to isolate a series of fractions containing progressively smaller lipoproteins. Successive fractions showed progressive enrichment in cholesteryl-esters, depletion of triglyceride, slower migration on agarose, and depletion of apo E and the C apolipoproteins relative to apo B. The IDL-1 and IDL-2 subspecies appeared to represent two distributions of lipoproteins overlapping in size (particle diameter 280-300 A and 268-284 A, respectively) and buoyant density (d = 1.008-1.022 g/ml and d = 1.013-1.028 g/ml, respectively). Plasma from two patients with dysbetalipoproteinemia showed predominant increases in fractions normally containing small VLDL and IDL-1, while patients with severe hypertriglyceridemia had increases primarily in levels of larger VLDL. Heterogeneity of lipoprotein precursors could account for the discrete subspecies of LDL observed in human plasma.

Interaction of human-plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine, and characterization of the interaction products

Interaction of human low-density lipoproteins (LDL) with discoidal complexes comprised of egg yolk phosphatidylcholine and human apolipoprotein A-I (molar ratio, 88:1, respectively) was investigated. The multicomponent gradient gel electrophoretic pattern of LDL is transformed to one that includes a predominant component with an apparent particle diameter larger than that of the initial major LDL but still in the size range of normal LDL. The apparent particle diameter increase (range, 0.2–3.5 nm) is proportional to the increase (range, 6–40%) in LDL phospholipid/protein weight ratio following incubation (37°C; 6 and 24 h); the smaller the initial LDL diameter, the greater the apparent particle diameter increase and percentage of phospholipid uptake. The LDL unesterified cholesterol/protein weight ratio decreases (range, 33–39%), but does not correlate with the increase in apparent particle diameter value. Interaction products are round particles with intact apolipoprotein B and show no evidence of phospholipid degradation. The products appear more dense than expected from the size vs. density relationship observed for nonincubated LDL subspecies. In addition to products in the normal LDL size range, larger components (apparent particle diameter range, 29.0–41.2 nm) also form and may be association complexes of phospholipid-modified LDL. Our results indicate that phospholipid uptake by LDL may contribute to the particle size polydispersity observed in plasma LDL.

Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells

Low density lipoprotein (LDL) subspecies of different size and lipid mass were isolated by density gradient ultracentrifugation from the serum of male rhesus monkeys ( Macaca mulatta) fed both a low fat, low cholesterol commercial primate ration, and cholesterol-supplemented high-fat diets, as well as from the serum of human donors. The mitogenic effect of these lipoproteins was examined using primary cultures of rhesus aortic smooth muscle cells. It was observed that the smaller LDL (molecular weight 2.7 × 10 6) from normolipidemic monkeys and a small LDL (molecular weight 2.6 × 10 6) occurring in some normal human subjects exhibited no mitogenic action. In turn, the larger LDL subspecies (molecular weight >3.0 × 10 6, and buoyant density <1.030 g/ml), whether from normolipidemic or hyperlipidemic monkeys, or from some normal human subjects, had a marked proliferative action. The results indicate that both hyperlipidemic and normal sera (both human and rhesus) contain mitogenic LDL species although in different amounts. LDL-III, the rhesus equivalent of human Lp(a) was not mitogenic despite its similarity in size and lipid composition to the stimulating particles. However, on the removal of most of its large sialic acid moiety, a clear mitogenic action was observed. The mechanisms responsible for the proliferative effect are unclear and may involve LDL mass, lipid composition, and surface charge although other speculations cannot at present be ruled out. Furthermore, since the small LDL subspecies of either rhesus or human origin were nonmitogenic and similar in mass to the LDL found in calf serum, the mitogenic response of the smooth muscle cells to large LDLs may depend on their early conditioning with the LDL of calf serum.

Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

Density gradient ultracentrifugation of low density lipoproteins (LDL) from 12 normal subjects showed multiple, distinct isopycnic bands. Densitometric scanning of the gradient tubes revealed that each band could be assigned to one of four density intervals and that the boundaries of these intervals were consistent among all the subjects. Analytic ultracentrifuge flotation (S(f)(0)) rates were assigned to the four density intervals, and there was a strong correlation between peak S(f)(0) rate and peak isopycnic banding position (R(f)) of the LDL in the 12 subjects. The S(f)(0) value corresponding to the boundary between the two most buoyant LDL density subgroups was 7.5. This value is close to that previously demonstrated to define two LDL subdivisions (S(f)(0) 0-7 and S(f)(0) 7-12) that were discriminated by differing concentrations in men and women, and differing statistical relationships with levels of HDL and VLDL in a normal population. Further delineation of distinct subspecies of LDL was afforded by electrophoresis in 2-16% gradient polyacrylamide gels. Densitometric scans of protein-stained gels revealed multiple peaks, and particle diameters were assigned to these peaks using calibration markers. Particles of diameter >/= 280 A included both IDL and Lp(a), the latter defined by pre-beta mobility on agarose electrophoresis and density > 1.050 g/ml. LDL particles with diameters 220-272 A could be grouped into seven size intervals defined by modes in the distribution of gradient gel electrophoretic peaks in LDL from a group of 68 healthy men and women. Particle diameters of the major peaks in each of seven density subfractions decreased with increasing density of the fractions. However, particles within each of the size groups were distributed across a range of densities. Use of a lipid-staining procedure allowed identification of electrophoretic bands in whole plasma which corresponded to those seen in isolated LDL, eliminating the possibility that ultracentrifugation was responsible for formation of the subspecies detected by the gradient gel procedure. The application of density gradient ultracentrifugation and gradient gel electrophoresis provides a means of characterizing LDL from normal humans in terms of multiple distinct subpopulations which may also prove to have differing metabolic and pathologic properties.-Krauss, R. M., and D. J. Burke. Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

Heterogeneity of human plasma low density lipoprotein

Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions.

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [ 14C]Ampholine, and gel filtration completely separated the lipoproteins from [ 14C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.