Characterization of plasma low density lipoproteins on nonhuman primates fed dietary cholesterol.

LDL from animals of three nonhuman primate species, Macaca mulatta, Macaca fascicularis, and Cercopithecus aethiops, were studied. A standard preparation of 125I-LDL was added to isolated lipoprotein mixtures just prior to separation of plasma lipoproteins by agarose gel chromatography. A relative size index, rI, was determined by dividing the elution volume of the iodinated LDL by the elution volume of the sample LDL, both volumes being determined simultaneously during chromatographic elution. Comparison of rI with molecular weights measured by flotation equilibrium analysis in the analytical ultracentrifuge showed a linear relationship across a molecular weight range of 2.5-8.0 X 10(6), r = 0.985. A regression equation describing this relationship was used to calculate molecular weights of LDL from a group of M. fascicularis that were fed cholesterol-containing diets. In these animals, plasma cholesterol concentration ranged from 100 to over 700 mg/dl and was highly correlated with LDL molecular weight and with the micromolar concentration of the LDL. Using multiple regression analyses, the two variables of plasma LDL could be shown to account for 94% of the variation in plasma cholesterol concentration in the M. fascicularis of this study. Micromolar concentration and molecular weight of LDL were not correlated with each other, suggesting that in M. fascicularis at least two independent types of controls are operative in the response of plasma LDL to dietary cholesterol. The increase in LDL molecular weight was associated with a large increase in cholesteryl ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. As molecular weight increased, these components appeared to be added to the LDL particles together as discrete increments of fixed composition. The data are consistent with a spherical model of LDL structure with a core of cholesteryl ester and triglyceride and a 21.3 A-thick coat of phospholipid, free cholesterol, and protein.     

A species comparison of low density lipoprotein heterogeneity in nonhuman primates fed atherogenic diets

Six male cynomolgus monkeys and five male African green monkeys were fed dietary cholesterol to induce hypercholesterolemia. The two groups studied had equivalent total plasma cholesterol concentrations. Low density lipoproteins (LDL) were isolated from whole plasma by ultracentrifugation and separated from other lipoprotein classes by agarose column chromatography. LDL were further subfractionated by density gradient ultracentrifugation in a VTi-50 vertical rotor. The material within five density regions was pooled from each sample and molecular weight, electrophoretic mobility, apoprotein heterogeneity, and percentage composition were determined for each subfraction. In general, cynomolgus monkey LDL were larger and more polydisperse than African green monkey LDL, and the LDL subfractions of cynomolgus monkeys were generally of lower densities although molecular weights at any density were in the same range for both species. ApoB-100 was the major apoprotein in each subfraction. ApoE was frequently present in the less dense subfractions while apoA-I was often seen in the more dense subfractions. Cynomolgus monkey LDL appeared to contain more apoE than African green monkey LDL. Over the entire spectrum of LDL, the percentage composition of the particles at any given density was indistinguishable between the species. In general, the average cynomolgus monkey LDL was larger, more polydisperse, less dense, and appeared to contain more apoE than the average African green monkey LDL. One or all of these differences might help explain the increased susceptibility to diet-induced atherosclerosis seen in cynomolgus monkeys.     

Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins.

The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.     

Comparison of apoprotein B of low density lipoproteins of human interstitial fluid and plasma.

Virtually all apoprotein B (apoB)-containing lipoproteins of the peripheral interstitial fluid of subjects with primary lymphoedema float in the ultracentrifugal field in the density interval 1.019-1.063 g/ml; in this respect they are similar to plasma low-density lipoproteins (LDL). 2. Virtually all apo-B-containing lipoproteins of interstitial fluid migrate in the electrophoretic field with pre-beta mobility; in this respect they are similar to plasma very-low-density lipoproteins. 3. The apoB of lipoproteins of interstitial fluid does not differ in terms of Mr from apoB-100 of human plasma [Kane, Hardman & Paulus (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2465-2469] as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. Both apoB of interstitial fluid and plasma are heterogenous in terms of their charge as determined by isoelectric focusing of their complexes with the nonionic detergent Nonidet P40. ApoB of plasma LDL focuses between pH5.9 and 6.65, and that of interstitial fluid LDL between pH 5.9 and 6.1. Thus the overall charge of apoB of interstitial fluid is more negative than that of its plasma LDL counterpart.     

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.     

Triacylglycerol and very low density lipoprotein secretion into plasma of squirrel monkeys.

We determined the effects of varying the types and level of dietary fat and cholesterol on the increase in plasma total triacylglycerol concentrations after injection of Triton WR-1339, an inhibitor of lipoprotein lipase, into monkeys that had been subjected to an overnight fast. The monkeys that had been treated with Triton WR-1339 were then given a test meal by intragastric intubation. Dietary cholesterol, high levels of fat and saturated fat in the habitual diet reduced the rate of release of triacylglycerol to plasma in the fasted monkey. We also determined the changes in protein and lipid concentrations of the different lipoprotein fractions. The injection of Triton WR-1339 resulted in a linear increase with time in the concentration of protein and triacylglycerol in the very low density (chylomicron-free and d less than 1.006) lipoproteins, but there was an increase in the ratio of traicylglycerol to protein in that fraction. Most of the increase (96%) in very low density protein was in the B protein. Regardless of the habitual diet, a test meal accentuated the rate of triacylglycerol appearance in whole plasma and in the very low density lipoproteins of Triton WR-1339-treated monkeys, and the rate of increase of the protein component after feeding was slightly higher. Thus the administration of a meal to the fasted Triton WR-1339-treated squirrel monkey further increased the proportion of triacylglycerol in very low density lipoproteins. Although dietary cholesterol and saturated fat in the habitual diet depressed the rate of increase in very low density triacylglycerol during fasting, the rate of protein synthesis was not significantly affected. After administration of a test meal the rates of increase in triacylglycerol and protein in the very low density lipoproteins were similar for monkeys from the different diet groups. Triton WR-1339 administration caused a slight and progressive increase in the intermediate density (d 1.006-1.019) lipoproteins and a marked and progressive decrease in the low density (d 1.019-1.063) lipoproteins. There was an immediate (by 5 min) drop of 70% or more in high density (d 1.063-1.21) lipoprotein protein, but the lipids except triacylglycerol remained unchanged. There was a decrease in both the A (the major fraction) and C proteins. The rates of very low density B protein secretion were comparable to the rates of low density lipoprotein catabolism that had been previously demonstrated for this species.     

1H NMR studies of lymph chylomicra and very low density lipoproteins from nonhuman primates

1H NMR spectroscopy at 200 MHz was used to study triglyceride crystalline leads to liquid transitions which occurred on heating between 10 and 50 degrees C in very low density lipoprotein and subfractionated chylomicron particles from nonhuman primates fed a saturated fat (butter fat) diet. Model system studies of pure triglycerides (triolein, tripalmitin and a 1:1 mixture) and emulsion particles consisting of these triglycerides with a surface of egg phosphatidylcholine showed that high resolution spectra were obtained only from liquid triglycerides. In lipoprotein spectra, changes in 1H NMR peak intensities and line widths accompanied the solid leads to liquid transition of the constituent triglycerides. Peak areas of fatty acyl resonances were proportional to the percentage of melted triglyceride determined by differential scanning calorimetry. NMR peak area measurements showed that the calorimetric transition involved the melting of relatively greater numbers of saturated fatty acyl chains than unsaturated chains; at temperatures well below the solid leads to liquid transition, the lipoproteins contained a significant fraction (approximately 33%) of liquid triglycerides which were relatively enriched in unsaturated fatty acyl chains. For model systems containing mixtures of solid and liquid triglycerides, the temperature dependence of line widths of fatty acyl resonances demonstrated that solid triglycerides decreased the mobility of the liquid triglycerides. A similar temperature dependence for the lipoprotein resonances suggested that solid and liquid species are co-mixed in individual lipoprotein particles within a purified subfraction. Temperature-dependent line width and intensity changes were observed for the phospholipid-choline methyl resonance in lipoprotein spectra and were apparently independent of the core transition.     

Hepatic perfusate very low density lipoproteins obtained from fat-fed nonhuman primates stimulate cholesterol esterification in macrophages

The livers of both baboons and rhesus monkeys fed a high fat, high cholesterol diet secreted very low density lipoproteins (VLDL) that were enriched in cholesteryl ester and apoe as compared to VLDL secreted by the livers of chow-fed animals. Stimulation of macrophage cholesterol esterification by the experimental VLDL was compared to that produced by the standard beta-VLDL obtained from the plasma of a rhesus monkey fed 25% coconut oil plus 2% cholesterol. This standard beta-VLDL stimulated 7- to 10-fold more esterification than did the bovine albumin control. Hepatic VLDL from fat-fed animals stimulated esterification in J774 macrophages 50 to 150% as well as did the standard beta-VLDL, even though hepatic VLDL did not display beta electrophoretic mobility on agarose gel electrophoresis. Plasma VLDL from lard-fed baboons did not exhibit beta electrophoretic mobility but did stimulate esterification in macrophages. Baboons were divided into high and low responders based on the change in plasma cholesterol levels in response to a high fat, high cholesterol diet. Both plasma and hepatic VLDL from high responders stimulated cholesterol esterification, whereas hepatic VLDL obtained from low responders or chow-fed baboons did not stimulate cholesterol esterification in macrophages. There was a strong positive correlation (r = 0.866) between the number of apoE molecules per VLDL particle in VLDL obtained from chow-fed, lard-fed, or coconut oil-fed primates and the rate of cholesterol esterification in macrophages. Our results show that hepatic perfusate VLDL obtained from fat- and cholesterol-fed primates have compositional and functional properties usually ascribed to circulating beta-VLDL, without displaying beta mobility, and indicate that the liver may be an important source of atherogenic lipoproteins.     

Studies on pig serum lipoproteins. VI. Surface charge of very low density lipoprotein.

The surface electric charge of pig serum very low density lipoprotein (VLDL) is described. By isoelectric focusing VLDL was separated into at least 3 fractions having different isoelectric points and polypeptide distributions. The ultracentrifugal and electron microscopic results indicate that the VLDL was not drastically denatured by Ampholine.     

Subunit structure of the apoprotein of human serum low density lipoproteins

Human serum low density lipoproteins (d 1.027–1.043 g/cm³) were prepared by preparative ultracentrifugation and delipidated with sodium deoxycholate. By electrophoresis in sodium dodecyl sulfate polyacrylamide gel, the apoprotein was fractionated into major components with apparent molecular weights of 77,000, 66,000, 47,000, 33,500, 21,500, 13,000, and 9,500, respectively; and minor components of higher molecular weight. The data indicate the existence of at least two fundamental subunits of molecular weights of approximately 9,500 and 13,000 daltons.     

Protective effect of lipoproteins containing apoprotein A-I on Cu2+-catalyzed oxidation of human low density lipoprotein

Two apoprotein A-I (apoA-I)-containing lipoproteins, one containing apoA-I and apoA-II (LpA-I/A-II) and the other containing only apoA-I (LpA-I), were examined for their effect on Cu2+-mediated oxidation of low density lipoprotein (LDL). The presence of LpA-I or LpA-I/A-II prevented LDL oxidation when assessed by the electrophoretic mobility, apoprotein B fragmentation and amounts of thiobarbituric acid-reactive substances. The protection of LDL oxidation by these lipoproteins was effective for up to 6 h, with LpA-I being more active than LpA-I/A-II. Results from these in vitro model experiments raise a possibility that LpA-I may play a role in protecting LDL from Cu2+-mediated oxidation.     

Endogenously labeled low density lipoprotein triglyceride and apoprotein B kinetics.

The kinetics of endogenously labeled low density lipoprotein (LDL) triglycerides (TG) and apoprotein B (apoB) have been studied in four normal and in four hyperlipemic subjects using double tracers. Analysis of the data suggests that most LDL triglycerides turn over about 10 times faster than apoB (0.003/min vs. 0.0003/min) and that about 10% of the LDL particles contain most of the TG found with LDL. It is not possible to determine from the analysis whether each new LDL particle arrives with the excess TG or whether only a subpopulation of particles contains most of the TG. The kinetic analysis further suggests that triglyceride-rich LDL particles do not exchange with an extraplasma compartment as most LDL particles do, and thus, they behave more like very low density lipoprotein particles. A compartmental model accounting for both the LDL-TG and LDL-apoB kinetics is proposed.