A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.     

Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins.

The relation of coronary artery disease to plasma lipoproteins was examined in 104 men aged 35-65 years undergoing coronary angiography for suspected myocardial ischaemia. A score reflecting the number, degree, and length of stenoses in seven major coronary arteries was assigned to each angiogram. Lipid concentrations in lipoprotein subfractions were measured after preparative ultracentrifugation; plasma apolipoprotein concentrations were measured by electroimmunoassay. Men with high coronary scores tended to have lower plasma high-density lipoprotein (HDL) cholesterol concentrations and higher low-density lipoprotein (density 1.019-1.063 g/ml) cholesterol concentrations than subjects of similar age with low coronary scores (p approximately equal to 0.1). The strongest relation, however, was with the cholesterol concentration in the HDL2 subfraction (density 1.063-1.125 g/ml) of HDL, which averaged 44% lower in the severely affected patients (p less than 0.005). No associations were found between the coronary score and HDL3 cholesterol, the cholesterol content of lipoproteins of density less than 1.019 g/ml, plasma triglyceride, or the concentrations of apolipoproteins AI, AII, and E. The high coronary scores associated with low HDL2 concentrations reflected an increase in the number of both partial and complete stenoses distributed throughout the coronary tree. In contrast the sizes of the lesions and the proportion producing complete occlusion were unrelated to HDL2.     

Catabolism of very low density lipoproteins in the rabbit. Effect of changing composition and pool size.

To determine the metabolic mechanism of hypercholesterolemia in rabbits produced by feeding cholesterol-rich diets, control and hypercholesterolemic rabbits were injected with I-labelled very low density lipoproteins (VLDL, d 1.006 g/ml) from control and/or hypercholesterolemic donors. Apolipoprotein B in VLDL decayed biphasically. The first phase occurred much more rapid than the second. 95% of the VLDL apolipoprotein B was catabolized via the first phase (t1/2 = 0.55 +/- 0.19 h) in normal rabbit with the immediate appearance of this radioactivity in intermediate density lipoproteins (IDL, d 1.006-1.025 g/ml) and low density lipoproteins (LDL, d 1.025-1.063 g/ml). The apolipoproteins C and E at the same time were transferred to high density lipoproteins where they decayed biphasically. The apolipoprotein B from hypercholesterolemic VLDL in the normal recipient disappeared at a similar rate as from normal VLDL via phase I; however, it was incompletely converted to IDL and LDL. Apolipoprotein B from normal VLDL in cholesterol-fed rabbits disappeared at a normal rate via phase I, but only 82% was catabolized by this phase. Hypercholesterolemic VLDL injected into the hypercholesterolemic recipient was less rapidly catabolized via phase I (T1/2 = 2.5 +/- 0.89 H) and only a small fraction was converted to IDL and LDL.     

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.     

Comparison of various methods for in vitro cholesteryl ester labeling of lipoproteins from hypercholesterolemic rabbits

Little or no information is available on biologically valid labeling of hypercholesterolemic plasma lipoproteins with cholesteryl ester. The esterification of labeled unesterified cholesterol in hypercholesterolemic rabbit plasma by the lecithin: cholesterol acyltransferase reaction is inefficient. The use of the d > 1.063 plasma fraction for this reaction greatly improves the efficiency, but some labeled unesterified cholesterol remains in the end products. The latter disadvantage can be avoided by the addition to whole plasma of labeled cholesteryl ester dissolved in DMSO or acetone. However, in hypercholesterolemic rabbit plasma only a small fraction of the added cholesteryl ester was associated with lipoproteins. When phosphatidylcholine/ cholesteryl ester liposomes were incubated with hypercholesterolemic rabbit plasma for 18–24 h at 37°C the labeled cholesteryl ester was quantitatively incorporated into lipoproteins. Chylomicron-like, cholesteryl ester-rich particles were removed by centrifugation (10⁶g · min) and the subsequently isolated d < 1.019 and d = 1.019–1.063 (LDL) fractions were injected intravenously into normal and hypercholesterolemic rabbits. The disappearance of d < 1.019 and LDL cholesteryl ester and the appearance of cholesteryl ester in other lipoprotein fractions was indistinguishable from that of in vivo-labeled lipoproteins. In vivo and in vitro cholesteryl ester-labeled lipoproteins were also compared by measuring the exchangeability of their cholesteryl ester with HDL cholesteryl ester in vitro. Equal exchangeability of the two labels was observed in the d < 1.019 fraction from which the chylomicron-like particles had been removed. These findings demonstrate that when cholesteryl ester is incorporated by the liposome procedure, the distribution of labeled cholesteryl ester within the lipoprotein complex corresponds closely to that of the in vivo-incorporated labeled cholesteryl ester.     

Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate

Although Triton WR 1339 has been used to block triglyceride or cholesterol removal from plasma, no data are available on the extent to which Triton WR 1339 administered to rabbits blocks clearance of newly absorbed dietary lipids. In the present study, we have measured the efficiency of this blockade during a 24-hr interval. After the Triton WR 1339 administration, plasma Sf greater than 400 and d less than 1.019 g/ml lipoprotein lipid concentrations increased greatly, but the concentration of d greater than 1.019 g/ml lipids decreased. In the rabbits fed 0.5% cholesterol for 1 week, the increase in d less than 1.019 g/ml and the decrease in 1.019 less than d less than 1.063 g/ml lipoprotein fractions 24 hr after the Triton WR 1339 injection were much greater than in the chow-fed Tritonized rabbits. After the Triton treatment, 50% of intravenously injected LDL-125I-labeled apoB disappeared in 24 hr, but little or no apoB appeared in other lipoprotein fractions and no VLDL apoB was converted to LDL. Labeled cholesterol and retinol were fed to rabbits and 24-hr increments in plasma cholesteryl- and retinyl-ester label and mass were measured. In chow-fed Tritonized rabbits about one-half of the absorbed oral doses of both labeled lipids was recovered in plasma, indicating that Triton WR 1339 does not completely inhibit the clearance of intestinal lipoproteins. When rabbits were injected with Triton and an ethyl oleate emulsion, the blockade of dietary lipid removal from plasma was substantially improved and chylomicron cholesterol uptake by extra-hepatic tissues was completely abolished.     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

Effects of cholestyramine on low density lipoprotein binding sites on liver membranes from rabbits with endogenous hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia with a lipoprotein distribution similar to that of humans. Approximately 76% of the total cholesterol is carried in the low density lipoprotein (LDL) fraction (1.006 less than d less than 1.063 g/ml). Inclusion of 1% cholestyramine in the diet prevents the increase in plasma cholesterol. The cholestyramine effect is mediated through an increased fractional catabolic rate of 125I-LDL. In order to determine the potential role of hepatic LDL receptors in the removal of LDL from the plasma, binding of 125I-LDL and 125I-beta-VLDL (beta-migrating very low density lipoproteins) to hepatic membranes prepared from livers of rabbits fed the wheat starch-casein diet with or without cholestyramine supplementation was investigated. Membranes from livers of the cholestyramine-supplemented animals exhibit high levels of specific EDTA-sensitive binding of either of the 125I-labeled lipoproteins. Very little EDTA-sensitive binding occurs on liver membranes from wheat starch-casein-fed rabbits that have not been treated with cholestyramine. These results indicate that the hypercholesterolemia in rabbits associated with the wheat starch-casein diet is wholly or partially the result of a decreased number of specific hepatic LDL receptors and thus a decreased catabolism of plasma cholesterol. The response of the liver to the inclusion in the diet of the bile acid sequestrant, cholestyramine, is to maintain or increase the number of specific LDL binding sites, thus promoting catabolism of plasma cholesterol.     

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.     

Serum lipoproteins of normal and cholesterol-fed rats

The density distribution of lipoproteins in rats fed chow or chow containing 1% cholesterol and 10% olive oil was studied. Lipoprotein fractions were prepared in the ultra-centrifuge between narrow density bands within the density range of 1.006-1.21 and were analyzed by chemical, electrophoretic, and immunological methods. In serum from normal rats there were three major lipoprotein fractions, with densities less than 1.006, 1.030-1.063, and 1.063-1.21. Almost no lipoprotein was found between d 1.006 and 1.030. Most of the low density lipoprotein appeared between a density of 1.04 and 1.05. In the density range 1.05-1.07, small amounts of both low density and high density lipoprotein were found. Feeding a diet high in cholesterol resulted in a marked increase in the concentration of lipoproteins of density less than 1.006, and a new lipoprotein fraction appeared between d 1.006 and 1.030; this fraction contained immunologically demonstrable low density and high density lipoproteins. In addition, there was a decrease in the high density lipoprotein fraction between d 1.070 and 1.21.     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

High density lipoprotein metabolism in normolipidemic and cholesterol-fed rabbits.

New Zealand white rabbits were used to determine the compositional and metabolic changes induced in high density lipoproteins (HDL, rho = 1.063--1.21 g/ml) in response to cholesterol feeding. There was no change in total HDL cholesterol in plasma due to cholesterol feeding (12 weeks), but the triglyceride level was decreased to 29% of pretreatment values. Total protein content of HDL was decreased in response to cholesterol feeding, resulting in a significant increase in the cholesterol/protein ratio. There was a decrease in some isomer of the major apolipoproteins (A-I2) of HDL. The decay of radioactivity in HDL or its apolipoproteins was biphasic in both normolipidemic and hypercholesterolemic rabbits. The first phase was much more rapid than the second. The decay rates for the radioactivity in HDL depended upon the dietary status of the recipient animal.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Physicochemical properties of low-density lipoproteins of normal human plasma. Evidence for the occurrence of lipoprotein B in associated and free forms

1. Low-density (d 1.006-1.063g/ml) lipoproteins from normal human plasma were separated by differential preparative ultracentrifugation into six subfractions. Each low-density (LD) lipoprotein subfraction contained lipoprotein B as the major and lipoproteins A and C as the minor lipoprotein families. 2. Three lipoprotein B subfractions (LP-B), LP-B-III (d 1.019-1.030g/ml), LP-B-IV (d 1.030-1.040g/ml) and LP-B-V (d 1.040-1.053g/ml) were prepared from the corresponding LD lipoprotein subfractions by immunoprecipitating small amounts of lipoproteins A and C. 3. Determination of hydrodynamic properties indicated that LD lipoproteins consisted of three molecular segments characterized by a stepwise change in the molecular weight: LDL-I and LDL-II subfractions (d 1.006-1.019g/ml) with an average mol.wt. of 4.75x10(6), LDL-III (d 1.019-1.030g/ml) with a mol.wt. of 3.99x10(6), and LDL-IV, LDL-V and LDL-VI (d 1.030-1.063g/ml) with a mol.wt. of 2.85x10(6). 4. All three lipoprotein B subfractions had an average mol.wt. of 3.16x10(6). 5. The LDL-I and LDL-II subfractions consisted of lipoprotein B and lipoprotein C families which were present in the form of an association complex. This was isolated from serum by immunoprecipitation with antibodies to lipoprotein B. The complex had a mol.wt. of 4.35x10(6). 6. The results indicate a fundamental difference between the LD lipoprotein subfractions with d 1.006-1.019g/ml and those subfractions with d 1.030-1.063g/ml. In the former, lipoprotein B occurs as a part of an association complex, whereas in the latter it occurs as a separate entity.     

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.     

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

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

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.     

Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.

Five glycosphingolipids (GSL), glucosylceramide, lactosylceramide, trihexosylceramide, globoside, and hematoside (GM3) were studied in serum from normal human subjects and patients with dyslipoproteinemia and found to be exclusively associated with the various classes of serum lipoproteins. Based on a unit weight of lipoprotein protein, the total amount of GSL in serum normal subjects was twice as high in very low density lipoprotein (VLDL) (d less than 1.006 g/ml) and low density lipoprotein (LDL) (d 1.019-1.063 g/ml) as in high density lipoproteins HDL2 (d 1.063-1.125 g/ml) or HDL3 (d 1.125-1.21 g/ml). In abetalipoproteinemia the levels of serum GSL were slightly reduced when compared to normal serum and were all found in the only existing lipoprotein, HDL; this contained 2-3 moles of GSL/ mole of lipoprotein as compared to 0.5 GSL/mole in normal HDL. In hypobetalipoproteinemia and Tangier disease, the serum glycosphingolipids were 10 to 30% reduced in concentration compared to the 75% reduction in other lipids, and were again found to be associated only with the serum lipoproteins. The relative proportions of GSL did not vary substantially in the normo- and hypolipidemic subjects studied. Only in patients with homozygous familial hypercholesterolemia was there a significant (3-4-fold) elevation of all of the five GSL species and this elevation of all of the five GSL species and this elevation correlated well with that of the circulating cholesterol and LDL. On a molar basis the LDL of these patients contained the same amount of GSL as normal subjects (5 moles GSL/mole protein). It is concluded that: (1) glycosphingolipids are associated only with the major lipoprotein classes in both normal and dyslipoproteinemic serum; (2) the relative proportions of the five glycosphingolipids are not significantly affected by dyslipoproteinemia; (3) only in severe hypolipoproteinemia do the remaining serum lipoproteins carry a complement of glycosphingolipids greater than normal. Although our results establish that glycosphingolipids are intimately associated with serum lipoproteins, the mode of association or the structural and functional significance of such an association remains undetermined.     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.