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.     

Density distribution and physicochemical properties of plasma lipoproteins and apolipoproteins in the goose, Anser anser, a potential model of liver steatosis

The fractionation and physicochemical characterization of the complex molecular components composing the plasma lipoprotein spectrum in the goose, a potential model of liver steatosis, are described. Twenty lipoprotein subfractions (d less than 1.222 g/ml) were separated by isopycnic density gradient ultracentrifugation, and characterized according to their chemical composition, particle size and particle heterogeneity, electrophoretic mobility, and apolipoprotein content. Analytical ultracentrifugal analyses showed high density lipoproteins (HDL) to predominate (approximately 450 mg/dl plasma), the peak of its distribution occurring at d approximately 1.090 g/ml (F1.21 approximately 2.5). The HDL class displayed marked density heterogeneity, HDL1-like particles being detected up to a lower density limit of approximately 1.020 g/ml, particle size decreasing progressively from 17-19 nm at d 1.024-1.028 g/ml to 10.5-12 nm (d 1.055-1.065 g/ml), and then remaining constant (approximately 9 nm) at densities greater than 1.065 g/ml. HDL subfractions displayed multiple size species; five subspecies were present over the range d 1.103-1.183 g/ml with diameters of 10.5, 9.9, 9.0, 8.2, and 7.5 nm, four in the range d 1.090-1.103 g/ml (diameters 10.5, 9.9, 9.0, and 8.2 nm) and three over the range d 1.076-1.090 g/ml (diameters 10.5, 9.9, and 9.0 nm). ApoA-I (Mr 25,000-27,000) was the major apolipoprotein in all goose HDL subfractions, while the minor components (apparent Mr 100,000, 91,000, 64,000, 58,000, approximately 42,000, 18,000 and apoC-like proteins) showed marked quantitative and qualitative variation across this density range (i.e., 1.055-1.165 g/ml). The d 1.063 g/ml boundary for separation of goose low density lipoproteins (LDL) from HDL was inappropriate, since HDL-like particles were present in the density interval 1.024-1.063 g/ml, while particles enriched in apoB (Mr approximately 540,000) and resembling LDL in size (approximately 20.5 nm) were detected up to a density of approximately 1.076 g/ml. Goose LDL itself was a major component of the profile (90-172 mg/dl) with a single peak of high flotation rate (Sf approximately 10.5). The physicochemical properties and apolipoprotein content of intermediate density lipoproteins (IDL) and LDL varied but little over the range d 1.013-1.040 g/ml, presenting as two particle species (diameters 20.5 and 21 nm) of essentially constant chemical composition; LDL (d 1.019-1.040 g/ml) were separated from HDL1 by gel filtration chromatography and appeared to contain primarily apoB with lesser amounts of apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Differences in carbohydrate content of low density lipoproteins associated with low density lipoprotein subclass patterns

The neutral carbohydrate content of both the protein (apoB) and lipid fractions of low density lipoproteins (LDL) from subjects with a predominance of small, dense LDL (subclass pattern B) was found to be lower than in subjects with larger LDL (subclass pattern A): 45 +/- 12 versus 64 +/- 13 mg/g apoLDL, and 58 +/- 8 versus 71 +/- 8 mg/g apoLDL (P less than 0.0005 for both). Sialic acid content of LDL lipids, but not apoB, was also reduced in subclass pattern B. ApoB and glycolipid carbohydrate content of total LDL and LDL density subfractions declined with increasing LDL density and decreasing particle diameter. Moreover, in LDL subfractions from pattern B subjects, carbohydrate content of LDL apoB, but not LDL glycolipid, was significantly lower in comparison with particles of similar size from pattern A subjects. Thus, in LDL subclass pattern B, reductions in LDL carbohydrate content are associated both with reduced concentrations of larger carbohydrate-enriched LDL subclasses, and with reduced glycosylation of apoB in all LDL particles. LDL glycolipids may vary with overall lipid content of LDL particles, but variation in apoB glycosylation may indicate differences in pathways for LDL production, and reduced apoB glycosylation may reflect the altered metabolic state responsible for LDL subclass pattern B.     

Physicochemical studies of two major subpopulations of low density lipoproteins by differential scanning calorimetry and n.m.r. spectroscopy

Two subpopulations, layer 2 (density 1.025-1.029 g/ml) and layer 3 (density 1.032-1.043 g/ml) of low density lipoproteins (LDL) were isolated from fresh human plasma of normal lipidaemic subjects by density gradient ultracentrifugation. Chemical analyses demonstrated the ratios of triglyceride/cholesterol ester decreased with increasing densities of subfractions. These subfractions together with triglyceride-rich lipoproteins (layer 1, density less than 1.019 g/ml) were subjected to physicochemical studies by differential scanning calorimetry (d.s.c.) and nuclear magnetic resonance (n.m.r.) spectroscopy. The average transition temperature (Tt) of layer 2 was 34.20 +/- 0.83 degrees C and that of layer 3 was 37.25 +/- 0.35 degrees C. In addition, many of the layer 3, but not layer 2 and layer 1, samples showed structural alteration and gave rise to an average Tt of 39.18 +/- 1.24 degrees C. The structural alteration could be detected with polarizing light microscopy showing birefringent spherulites at body temperature. The peak Tt values obtained by d.s.c. were in good agreement with those by n.m.r. spectroscopy. These results demonstrate the physicochemical heterogeneity within the LDL density region and suggest that layer 3 subpopulation is much more labile than the others.     

A density gradient study of the lipoprotein and apolipoprotein distribution in the chicken, Gallus domesticus

Plasma lipoproteins from 5-week old male chickens were separated over the density range 1.006-1.172 g/ml into 22 subfractions by isopycnic density gradient ultracentrifugation, in order to establish the distribution of these particles and their constituent apolipoproteins as a function of density. Lipoprotein subfractions were characterized by electrophorectic, chemical and morphological analyses, and their protein moieties were defined according to net charge at alkaline pH, molecular weight and isoelectric point. These analyses have permitted us to reevaluate the density limits of the major chicken lipoprotein classes and to determine their main characteristics, which are as follows: (1) very-low-density lipoproteins (VLDL), isolated at d less than 1.016 g/ml, were present at low concentrations (less than 0.1 mg/ml) in fasted birds; their mean diameter determined by gradient gel electrophoresis and by electron microscopy was 20.5 and 31.4 nm respectively; (2) as the the density increased from VLDL to intermediate density lipoproteins (IDL), d 1.016-l.020 g/ml) and low-density lipoproteins (LDL, d 1.020-1.046 g/ml), the lipoprotein particles contained progressively less triacylglycerol and more protein, and their Stokes diameter decreased to 20.0 nm; (3) apolipoprotein B-100 was the major apolipoprotein in lipoproteins of d less than 1.046 g/ml, with an Mr of 350000; small amounts of apolipoprotein B-100 were detectable in HDL subfractions of d less than 1.076 g/ml; urea-soluble apolipoproteins were present in this density range as minor components of Mr 38000-39000, 27000-28000 (corresponding to apolipoprotein A-1) and Mr 11000-12000; (4) high density lipoprotein (HDL, d 1.052-1.130 g/ml) was isolated as a single band, whose protein content increased progressively with increase in density; the chemical composition of HDL resembled that of human HDL2, with apolipoprotein A-1 (M 27000-28000) as the major protein component, and a protein of Mr 11000-12000 as a minor component; (5) heterogeneity was observed in the particle size and apolipoprotein distribution of HDL subfractions: two lipoprotein bands which additional apolipoproteins of Mr 13000 and 15000 were detected. These studies illustrate the inadequacy in the chicken of the density limits applied to fractionate the lipoprotein spectrum, and particularly the inappropriateness of the 1.063 g/ml density limit as the cutoff for LDL and HDL particle populations in the species.     

Physico-chemical properties of low density lipoproteins isolated in an equilibrium density gradient

The tryptophanyls of total low density lipoproteins (LDL) (1.006-1.063 g/ml) from coronary heart disease (CHD) patients and subjects without CHD signs had different accessibility to fluorescence quenchers (I-and acrylamide). LDL were separated into subfractions in equilibrium density gradient. The coefficient of extinction , quantum yield and other spectral characteristics of LDL intrinsic fluorescence, rotational mobility of maleimide spin labels and fatty acid spin probe were different in LDL subfractions from healthy subjects. LDL subfractions with hydrated density 1.045-1.05 g/ml bound to B,E-receptors of cultured fibroblasts more effectively than did subfractions with density 1.01-1.03 g/ml. Structural differences of apo-B in the particles with different lipid to protein ratio are supposed.     

Human low density lipoprotein subfractions separated by gradient gel electrophoresis: composition, distribution, and alterations induced by cholesteryl ester transfer protein

Low density lipoprotein (LDL) subfractions were studied in sera from 208 normolipidemic, 22 hypercholesterolemic, and 33 hypertriglyceridemic subjects. Whole serum without preliminary ultracentrifugation was submitted to electrophoresis in a nondenaturing polyacrylamide gel. Three main LDL patterns were observed in normolipidemic sera: type 1, characterized by the presence of only one major band; type 2, characterized by the presence of two close major bands; and type 3, where LDL were more dispersed and presented at least three distinct bands. Type 1 was more frequent in men (43%) than in women (19%). The tendency for a higher potential coronary disease risk profile sera, namely higher triglyceride level, higher very low density lipoprotein + LDL fraction and lower high density lipoprotein (HDL) fraction, was type 3 less than type 2 less than type 1. The LDL patterns found in hypercholesterolemic sera were of type 1. Hypertriglyceridemic sera were characterized by the presence of a major band of small size. Separated LDL subfractions were collected by electroelution and analyzed for composition. In all subspecies, the mass ratio of core to surface components was constant as well as the molar ratio of the two lipid surface components, phospholipids and free cholesterol. Surface lipid to apolipoprotein B ratio, cholesteryl ester to triglyceride ratio, and cholesteryl ester to apoB ratio increased with particle size increment. Incubation of LDL with HDL and purified cholesteryl ester transfer protein induced a transfer of lipids, mainly cholesteryl esters and phospholipids, to LDL and an increase of the sizes of LDL subfractions. This suggests that lipid transfers from HDL to LDL might be a process of intravascular LDL remodeling and a factor of LDL polymorphism.     

Characterization of equine plasma lipoproteins after separation by density gradient

1. Plasma lipoproteins from six thoroughbred horses were separated by density gradient ultracentrifugation. For each sample, lipoprotein bands were visualized by means of a prestained plasma control and characterized by electrophoretic, chemical and morphological analysis. 2. Very low density lipoproteins (VLDL) were isolated at d less than 1.018 g/ml. 3. Two clearly resolved bands were detected in the low density lipoprotein fraction (LDL). The density limits were evaluated as follows: LDL1(1.028 less than d less than 1.045 g/ml) and LDL2(1.045 less than d less than 1.070 g/ml). Marked differences were observed in the chemical composition and particle size of LDL1 and LDL2 fractions. 4. High density lipoprotein fraction (HDL) was usually isolated as a single band, distributed over the range 1.075 less than d less than 1.180 g/ml. However, chemical composition and particle size revealed heterogeneity in HDL subfractions. 5. The density limit of LDL and HDL bands varied in each animal, indicating differences in equine lipoprotein distribution.     

Size transformations of intermediate and low density lipoproteins induced by unesterified fatty acids

Plasma from individual human subjects is known to contain multiple discrete subpopulations of low (LDL) and intermediate (IDL) density lipoproteins that differ in particle size and density. The metabolic origins of these subpopulations are unknown. Transformation of IDL and larger LDL to smaller, denser LDL particles had been postulated to occur as a result of the combined effects of triglyceride hydrolysis and lipid transfer. However, the presence of multiple small LDL subspecies has been described in patients lacking cholesteryl ester transfer protein. We have characterized an alternative pathway in which size decrements in IDL or LDL are produced in the presence of unesterified fatty acids and a source of apolipoprotein (apo) A-I. Incubation of IDL or LDL subfractions with palmitic acid and either high density lipoproteins (HDL), apoHDL, or purified apoA-I gives rise to apoA-I, apoB-containing complexes that can dissociate into two particles, an apoB-containing lipoprotein with particle diameter 10-30 A smaller than the starting material, and a still smaller species (apparent peak particle diameter 140-190 A) containing lipid and apoA-I but no apoB. The newly formed IDL or LDL are depleted in phospholipid and free cholesterol with no change in apoB-100 as assessed by SDS gel electrophoresis. We hypothesize that this reaction may contribute to the formation of discrete IDL and LDL subpopulations of varying size during the course of hydrolysis of triglyceride-rich lipoproteins in plasma.     

Plasma lipoproteins and apolipoproteins in the preruminant calf, Bos spp: density distribution, physicochemical properties, and the in vivo evaluation of the contribution of the liver to lipoprotein homeostasis

The in vivo role of the liver in lipoprotein homeostasis in the preruminant calf, a functional monogastric, has been evaluated. To this end, the hydrodynamic and physicochemical properties, density distribution, apolipoprotein content, and flow rates of the various lipoprotein particle species were determined in the hepatic afferent (portal vein and hepatic artery) and efferent (hepatic vein) vessels in fasting, 3-week-old male preruminant calves. Plasma lipoprotein profiles were established by physicochemical analyses of a series of subfractions isolated by isopycnic density gradient ultracentrifugation. Triglyceride-rich very low density lipoproteins (VLDL) (d less than 1.018 g/ml) were minor plasma constituents (approximately 1% or less of total d less than 1.180 g/ml lipoproteins). The major apolipoproteins of VLDL were apoB-like species, while the complement of minor components included bovine apoA-I and apoC-like peptides. Particles with diameters (193-207 A) typical of low density lipoproteins (LDL) were present over the density interval 1.026-1.076 g/ml; however, only LDL of d 1.026-1.046 g/ml were present as a unique and homogeneous size subspecies, containing the two apoB-like species as major protein components in addition to elevated cholesteryl ester contents. LDL represented approximately 10% of total d less than 1.180 g/ml lipoproteins in fasting plasma from all three hepatic vessels. Overlap in the density distribution of particles with the diameters of LDL and of high density lipoproteins (HDL) occurred in the density range from 1.046 to 1.076 g/ml; these HDL particles were 130-150 A in diameter. HDL were the major plasma particles (approximately 90% of total d less than 1.180 g/ml substances) and presented as two distinct populations which we have termed light (HDLL) and heavy (HDLH) HDL. Light HDL (d 1.060-1.091 g/ml) ranged in size from 120 to 140 A, and were distinguished by their high cholesteryl ester (29-33%) and low triglyceride (1-3%) contents; apoA-I was the principal apolipoprotein. Small amounts of apolipoproteins with Mr less than 60,000, including apoC-like peptides, were also present. Heavy HDL (d 1.091-1.180 g/ml) accounted for almost half (47%) of total calf HDL, and like HDLL, were also enriched in cholesteryl ester and apoA-I; they ranged in size from 93 to 120 A. The protein moiety of HDLH was distinct in its possession of an apoA-IV-like protein (Mr 42,000). Blood flow rates were determined by electromagnetic flowmetry, thereby permitting determination of net lipoprotein balance across the liver. VLDL were efficiently removed during passage through the liver (net uptake 1.06 mg/min per kg body weight).(ABSTRACT TRUNCATED AT 400 WORDS)     

Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Chemical and physical properties.

Subfractions of CLDL (VLDL), Sf 100-400; CLDL2, Sf 60--100; VLDL3, Sf 20--60) and LDL (LDL), Sf 12--20; LDL2, Sf 6--12; LDL3, Sf 3--6) were isolated from the plasma of three normal, three type III and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies were of normal composition but were increased in concentration in the order VLDL1 greater than VLDL2 greater than VLDL3. In the same subjects, although LDL2 was lowered and LDL3 increased, the total plasma LDL concentration was normal. All VLDL subfractions were elevated in the type III group, but in this case VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL1 and was attributed to a substantial alteration in the cholesteryl ester : triglyceride ratio in the particle. A similar argument was used to explain thction in normal or type IV subjects. Particle diameters, determined by laser light-scattering spectroscopy were in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship between the physical and metabolic properties of VLDL and LDL subfractions in type III and IV hyperlipoproteinemia.     

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.     

Is hypertriglyceridemic very low density lipoprotein a precursor of normal low density lipoprotein?

The precursor-product relationship of very low density (VLDL) and low density lipoproteins (LDL) was studied. VLDL obtained from normal (NTG) and hypertriglyceridemic (HTG) subjects was fractionated by zonal ultracentrifugation and subjected to in vitro lipolysis. The individual subfractions and their isolated lipolysis products, as well as IDL and LDL, were rigorously characterized. A striking difference in the contribution of cholesteryl ester to VLDL is noted. In NTG subfractions, the cholesteryl ester to protein ratio increases with decreasing density (VLDL-I----VLDL-III). This is the expected result of triglyceride loss through lipolysis and cholesteryl ester gain through core-lipid transfer protein action. In HTG subfractions there is an abnormal enrichment of cholesteryl esters that is most marked in VLDL-I and nearly absent in VLDL-III. Thus, the trend of the cholesteryl ester to protein ratios is reversed, being highest in HTG-VLDL-I and lowest in VLDL-III. This is incompatible with the precursor-product relationship described by the VLDL----IDL----LDL cascade. In vitro lipolysis studies support the conclusion that not all HTG-VLDL can be metabolized to LDL. While all NTG subfractions yield products that are LDL-like in size, density, and composition, only HTG-VLDL-III, whose composition is most similar to normal, does so. HTG VLDL-I and VLDL-II products are large and light populations that are highly enriched in cholesteryl ester. We suggest that this abnormal enrichment of HTG-VLDL with cholesteryl ester results from the prolonged action of core-lipid transfer protein on the slowly metabolized VLDL mass. This excess cholesteryl ester load, unaffected by the process of VLDL catabolism, remains entrapped within the abnormal particle. Therefore, lipolysis yields an abnormal, cholesteryl ester-rich product that can never become LDL.     

Abnormalities of low density lipoproteins in normolipidemic type II diabetic and nondiabetic patients with coronary artery disease.

The characteristics of low density lipoproteins (LDL) of ten non-insulin-dependent diabetic (NIDDM) and ten nondiabetic patients with coronary artery disease (CAD) were investigated and compared to LDL of ten NIDDM patients without CAD and ten healthy persons. All subjects had LDL cholesterol below 160 mg/dl and serum triglycerides below 200 mg/dl. The mean LDL particle size and particle distribution profiles were analyzed by using nondenaturing polyacrylamide gradient gel electrophoresis. The LDL composition and hydrated density distribution were investigated by using density gradient ultracentrifugation. Both NIDDM and nondiabetic CAD patients tended to have larger LDL particles than NIDDM patients without CAD and healthy subjects. The increase of LDL particle size of CAD patients was due to marked enrichment of triglycerides (TG) in their LDL. The percentage content of TG in LDL of NIDDM patients with CAD was 14.5% and in LDL of nondiabetic CAD patients 13.4% compared with 7.9% in LDL of NIDDM patients without CAD and 7.2% in normal-LDL (P less than 0.05 or less between either CAD group and NIDDM without CAD or normals). The LDL TG/apolipoprotein (apo) B weight ratio was significantly higher in both CAD groups compared with LDL of the two groups without CAD (0.70 and 0.68 vs. 0.38 and 0.34, respectively, P less than 0.05, P less than 0.05 and P less than 0.01, P less than 0.01). The LDL total lipid to apoB weight ratio was similar in all four groups. Consistent with this, the hydrated density distributions of LDL in the four groups were similar, the average peak densities being 1.0346 g/ml, 1.0331 g/ml, 1.0331 g/ml, and 1.0331 g/ml, respectively. The findings of this study demonstrate that normolipidemic patients with CAD may have marked abnormalities in th eir LDL composition and these anomalies are present in both diabetic and nondiabetic patients.     

Alterations in plasma lipoproteins and apolipoproteins associated with estrogen-induced hyperlipidemia in the laying hen

The laying hen represents a physiological model in which the mechanisms of action of estrogens on lipid transport can be evaluated. The plasma lipoproteins in the laying hen were subfractionated into discrete particle species by isopycnic density gradient ultracentrifugation and the physicochemical properties and apolipoprotein contents of individual subfractions evaluated. The qualitative and quantitative aspects of this estrogen-specific profile were then compared to those of the immature chicken. As observed earlier, estrogens induced dramatic elevation in very-low-density lipoproteins (VLDL) (up to 900 mg/dl). Indeed, triglyceride-rich lipoproteins with densities up to 1.035 g/ml, i.e. VLDL and their remnants, behaved as a continuum which displayed little variation in size (20.5-21 nm), electrophoretic mobility (beta-like) and apolipoprotein content; apo B-100 (540 kDa) predominated while apo A-I (27 kDa), apo VLDL-II (19 kDa) and an apo-C-like protein (13 kDa) were present as minor components. The typical high-density lipoproteins (HDL) in the immature chicken were replaced by a lipoprotein population whose physicochemical properties were quite distinct. Thus these particles were distributed as a single, asymmetric peak over the density range 1.030-1.158 g/ml, a wide interval which overlapped that of apo-B-rich particles at its lower limit. The rho 1.030-1.158 g/ml lipoproteins were present at concentrations (approximately equal to 200 mg/dl) some twofold to threefold lower than those of HDL in immature birds. Furthermore, they displayed physical and chemical properties in common with both low-density lipoproteins (LDL) and HDL and were LDL-like in exhibiting beta mobility but HDL-like in size (9-15 nm diameter). Their protein moiety was also HDL-like in its predominant content of apo A-I; small amounts of apo VLDL-II and the apo-C-like protein were also detected. Substantial amounts of lipid were found at rho greater than 1.195 g/ml: such substances are absent in the immature chicken and may reflect the presence of vitellogenins. The hyperestrogenic state in the laying hen is therefore associated with major modifications in lipoprotein and apolipoprotein profile. Such modifications may be of relevance to clinical disorders involving estrogen-induced hyperlipidemia.     

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 g/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 g protein HDL/ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.     

Fractionation of human low density lipoprotein by column chromatography.

We have devised a method to fractionate low density lipoprotein (LDL) into subspecies by means of column chromatography. DEAE-agarose columns, 2.6 X 60 cm, were loaded with LDL (25-45 mg LDL protein) and eluted with a 0.045-0.13 M NaCl gradient. The LDL eluted over a volume of 900 ml. Specific portions of the eluted LDL, reapplied to a column identical with the original, reelute at about the same point. Altering the NaCl concentration of the elution fluid changed the elution volume. The cholesterol-protein ratio of the LDL subfractions was progressively lower in fractions eluting at higher NaCl concentrations. These results indicate the LDL is not a homogenous lipoprotein species but consists of subfractions which differ in at least charge and cholesterol content.