Evaluation of gel chromatography for plasma lipoprotein fractionation

The fractionation of lipoproteins of normal and hyperlipidemic subjects on a column of 2% agarose was compared with ultracentrifugation and paper electrophoresis procedures. The following results were obtained. (a) Plasma lipoproteins were eluted successively from the column in the four overlapping peaks of chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins. (b) Very low density lipoproteins and high density lipoproteins (d > 1.063, containing nonlipoprotein proteins) showed continuous progressive changes in lipid composition as these fractions emerged, while low density lipoproteins showed a relatively constant lipid composition. (c) A discontinuous transition of lipid composition was observed when consecutive ultracentrifugal fractions were placed on the column. (d) The "trail" of pre-beta lipoprotein seen on paper electrophoresis was shown to consist of particles whose molecular sizes range between chylomicrons and pre-beta lipoproteins. A reverse relationship was observed between electrophoretic mobilities of "trail" components and their particle size. (e) Gel with an agarose content of 2% seemed to fractionate chylomicrons and very low density lipoproteins more effectively than other lipoprotein classes.     

In vitro incorporation of cholesterol-14C into very low density lipoprotein cholesteryl esters

The cholesteryl esters of very low density lipoproteins become labeled when human plasma is incubated with cholesterol-(14)C. The relative order of magnitude of the specific activity of the cholesteryl esters of the major lipoprotein fractions is: high density lipoproteins > very low density lipoproteins > low density lipoproteins. This pattern of labeling is similar to that found by others in experiments performed in vivo. Very low density lipoprotein cholesteryl esters are probably not formed by direct action of the plasma lecithin:cholesteryl acyltransferase, since significant esterification of cholesterol does not occur when very low density lipoproteins are incubated separately with the enzyme. Instead, labeled cholesteryl esters formed in the other lipoprotein fractions transfer to the very low density lipoproteins, the relative amount of monounsaturated esters transferred being slightly greater than that of saturated and polyunsaturated esters. The results support the possibility that the acyltransferase indirectly increases the concentration of very low density lipoprotein cholesteryl esters in vivo.     

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.     

A new large chylomicron remnant from cholesterol-fed rabbits

The lipoproteins of density less than 1.063 g/ml of cholesterol-fed rabbits were subjected to analytical ultracentrifugation. In many rabbits two peaks were found in the very low density (Sf greater than 20) portion of the lipoprotein spectrum. They were isolated by preparative ultracentrifugation and analysed. The smaller particles (remnant chylomicrons) had a peak Sf of 37, mean diameter of 36 nm, mean density of 1.00 g/ml, and their chemical composition agreed closely with previous reports. The larger particles had a peak Sf of 270, mean diameter of 80 nm, mean density of 0.97 g/ml and a high (80%) cholesterol ester and low (4%) triglyceride content. The fatty acid composition of the cholesterol esters, phospholipids and triglycerides was similar in both fractions. It is proposed that these large lipoprotein particles are also remnant chylomicrons. Possible reasons are presented to explain the presence of this second peak in the very low density lipoprotein spectrum.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

Effects of substrate composition on the esterification and hydrolysis activity of lysosomal acid sterol ester hydrolase

Lipid microemulsions with various core and surface lipid compositions were prepared by co-sonication of cholesteryl esters, triolein (TO), egg phosphatidylcholine (egg PC), and cholesterol. The heterogeneous emulsion particle mixture was purified by gel filtration and particles with the size and general organization of low density lipoproteins were obtained. These lipid microemulsion particles were used for studies of the cellular metabolism of lipoprotein-derived cholesterol and cholesteryl esters as catalyzed by the enzyme acid sterol ester hydrolase (EC 3.1.1.13). The hydrolysis of cholesteryl oleate (CO) was more than twice and that of cholesteryl linoleate (CL) more than three times faster than the hydrolysis of cholesteryl stearate (CS) over the temperature range 25-39.6 degrees C. Both the synthesis and hydrolysis of cholesteryl esters were insensitive to the physical state of the microemulsion cores. The synthesis of cholesteryl esters by this enzyme was also insensitive to the ratios of cholesterol and egg PC in the microemulsion surface layers. Incorporation of triolein into the microemulsion cholesteryl ester core slightly increased the rate of cholesteryl ester synthesis. A decreasing fatty acyl chain length (C18:0 to C14:0) and an increasing degree of unsaturation (C18:0 to C18:2) enhanced the synthesis rate. It is suggested that the hydrolysis and synthesis of cholesteryl esters in microemulsions (and lipoproteins) take place only in the particle surface layer and that the rate of catalysis is directly dependent on the amount of substrate in this surface layer.     

Characterization and catabolism of rat very high density lipoproteins

A previously unrecognized lipoprotein of very high density was isolated from rat serum. During zonal ultracentrifugation of whole serum or of fractions from Sepharose 4B chromatography, a peak comigrating with a peak of cholesterol was found between the typical high density lipoproteins and the residual serum proteins. Centrifugation of chylomicrons, very low density lipoproteins, and high density lipoproteins, radio-iodinated in their lipid and protein moieties and mixed with serum, did not yield this peak. The pooled fractions contained about 85% protein. The remainder was lipid comprising cholesteryl esters, free cholesterol, triglycerides, phosphatidylcholine, and sphingomyelin. Polyacrylamide gel electrophoresis revealed bands in the region of apolipoproteins E and C as the major components. The composition suggested a lipoprotein, and this was substantiated by electron microscopy which showed particles with a mean diameter of 150 A. Their average hydrated density was 1.23 g/ml and the apparent molecular weight was 1.35 X 10(6). These very high density lipoproteins are characterized by a rapid catabolism as compared to high density lipoproteins. Within 10 min, 84% and 70% of intravenously injected 125I-labeled very high density lipoproteins were removed from plasma of male and female rats, respectively, and did not appear to be converted to lipoproteins of a different density class. Ninety-five percent of the removed 125I was recovered in the liver and the radioactivity per gram of tissue was also highest for the liver. Accordingly, the rate of clearance of 125I-labeled very high density lipoproteins was markedly reduced in functionally eviscerated rats. Radioautography revealed that most of the silver grains representing very high density lipoproteins were associated with hepatocytes and only about 1% was found over v. Kupffer cells. Uptake and degradation by freshly isolated rat hepatocytes were mediated by a saturable and specific binding site. Composition and metabolic pathway are compatible with a function of very high density lipoproteins in the transport of protein and lipids to the liver.     

Qualitative and quantitative alterations of bovine serum lipoproteins with ageing

1. Plasma lipoproteins from six calves at 8, 43 and 118 days old, six heifers and six cows were separated by density gradient ultracentrifugation. For each sample lipoproteins bands were visualized by prestained control and characterized by electrophoretic, chemical and morphological analysis. 2. Two resolved bands were detected in the low density lipoprotein fraction (LDL). At an early stage of development, LDLI and LDLII were present with almost equal concentration. With ageing, LDLII became the major fraction of LDL lipoproteins. 3. HDL were isolated as a single band distributed over the range 1.064-1.166 mg/ml in young calf and 1.050-1.152 mg/ml in adult. This progressive decrease of density limits with ageing, associated with a decrease of protein content and an increase of phospholipids and cholesteryl esters content, was consistent with higher HDL particle diameters in adult. 4. With ageing, free cholesterol/esterified cholesterol ratio decreased in LDL fractions and increased in HDL fractions.     

Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin

The net transfer of core lipids between lipoproteins is facilitated by cholesteryl ester transfer protein (CETP). We have recently documented CETP deficiency in a family with hyperalphalipoproteinemia, due to a CETP gene splicing defect. The purpose of the present study was to characterize the plasma lipoproteins within the low density lipoprotein (LDL) density range and also the cholesteryl ester fatty acid distribution amongst lipoproteins in CETP-deficient subjects. In CETP deficiency, the conventional LDL density range contained both an apoE-rich enlarged high density lipoprotein (HDL) (resembling HDLc), and also apoB-containing lipoproteins. Native gradient gel electrophoresis revealed clear speciation of LDL subclasses, including a distinct population larger in size than normal LDL. Anti-apoB affinity-purified LDL from the CETP-deficient subjects were shown to contain an elevated triglyceride to cholesteryl ester ratio, and also a high ratio of cholesteryl oleate to cholesteryl linoleate, compared to their own HDL or to LDL from normal subjects. Addition of purified CETP to CETP-deficient plasma results in equilibration of very low density lipoprotein (VLDL) cholesteryl esters with those of HDL. These data suggest that, in CETP-deficient humans, the cholesteryl esters of VLDL and its catabolic product, LDL, originate predominantly from intracellular acyl-CoA:cholesterol acyltransferase (ACAT). The CETP plays a role in the normal formation of LDL, removing triglyceride and transferring LCAT-derived cholesteryl esters into LDL precursors.     

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.     

Preferential enrichment of large-sized very low density lipoprotein populations with transferred cholesteryl esters

The effect of lipid transfer proteins on the exchange and transfer of cholesteryl esters from rat plasma HDL2 to human very low (VLDL) and low density (LDL) lipoprotein populations was studied. The use of a combination of radiochemical and chemical methods allowed separate assessment of [3H]cholesteryl ester exchange and of cholesteryl ester transfer. VLDL-I was the preferred acceptor for transferred cholesteryl esters, followed by VLDL-II and VLDL-III. LDL did not acquire cholesteryl esters. The contribution of exchange of [3H]cholesteryl esters to total transfer was highest for LDL and decreased in reverse order along the VLDL density range. Inactivation of lecithin: cholesterol acyltransferase (LCAT) and heating the HDL2 for 60 min at 56 degrees C accelerated transfer and exchange of [3H]cholesteryl esters. Addition of lipid transfer proteins increased cholesterol esterification in all systems. The data demonstrate that large-sized, triglyceride-rich VLDL particles are preferred acceptors for transferred cholesteryl esters. It is suggested that enrichment of very low density lipoproteins with cholesteryl esters reflects the triglyceride content of the particles.     

Effects of the degree of saturation of dietary fat on the hepatic production of lipoproteins in the African green monkey

The cholesteryl ester content of plasma low density lipoproteins (LDL) in monkeys has previously been shown to be related to the rate of hepatic cholesterol secretion and cholesteryl ester content of newly secreted lipoproteins in the isolated perfused liver. In the present studies, African green monkeys were fed diets containing cholesterol and 40% of calories as either butter or safflower oil in order to determine the effects of saturated versus polyunsaturated dietary fat on hepatic lipoprotein secretion. The rate of cholesterol accumulation in liver perfusates was correlated with the size of the donor's plasma LDL, but for any rate, a smaller plasma LDL was found in donor animals of the safflower oil group than in those of the butter group. Hepatic very low density lipoproteins (VLDL) were smaller in the safflower oil group but contained more cholesteryl ester and fewer triglyceride molecules per particle than those from the butter group. Livers from the safflower oil group contained more cholesteryl ester and less triglyceride than those from the butter group. The cholesteryl ester percentage composition of hepatic VLDL resembled that of the liver in each group. The data show that dietary polyunsaturated fat decreased plasma LDL size even though it increased the cholesteryl ester content of lipoproteins secreted by the liver. Therefore, intravascular formation of plasma LDL from hepatic precursor lipoproteins appears to include the removal of relatively greater amounts of cholesteryl esters from the precursor lipoproteins in polyunsaturated fat-fed animals.     

Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Nascent high density lipoproteins from liver perfusates of orotic acid-fed rats

Uniformly fatty livers from orotic acid-fed rats secreted almost no very low density lipoproteins (VLDL) but normal amounts of nascent high density lipoproteins (HDL) accumulated in perfusates. When lecithin:cholesterol acyltransferase (LCAT) was inhibited, nascent HDL were uniformly discoidal and lacked cholesteryl esters. Lipid and apoprotein compositions of nascent HDL from normal and fatty livers were similar whether LCAT was inhibited or not. Apolipoprotein B-100 was not detected in perfusates of uniformly fatty livers, but small amounts of apolipoprotein B-48 were present in HDL2 fractions. Nascent lipoproteins were not seen in Golgi compartments, but lipid-rich particles were clearly evident in endoplasmic reticulum cisternae adjacent to the cis face of the Golgi complex, suggesting that orotic acid blocks VLDL secretion by preventing translocation of nascent particles from the endoplasmic reticulum to the cis Golgi compartment. The accumulation of normal amounts of discoidal HDL in liver perfusates despite virtual absence of triglyceride-rich lipoproteins in Golgi secretory compartments, the space of Disse, and the perfusate is inconsistent with the concept that nascent HDL are exclusively a product of surface remnants cast off during lipolysis of chylomicrons and VLDL.     

Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia.

Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.     

Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

Emulsions with lipid compositions similar to the triacylglycerol-rich lipoproteins were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. Radioactive labels tracing the emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the blood stream, but the removal rate of triacylglycerols was faster than that of cholesteryl ester. Most of the removed cholesteryl ester label was found in the liver, but only a small fraction of the triacylglycerol label was found in this organ, consistent with hepatic uptake of the remnants of the injected emulsion. Emulsions otherwise identical but excluding unesterified cholesterol were metabolized differently. The plasma removal of triacylglycerols remained fast, but the cholesteryl esters were removed very slowly. Heparin stimulated lipolysis, but failed to increase the rate of removal of cholesteryl esters from emulsions lacking cholesterol. Evidently, emulsions lacking cholesterol were acted on by the enzyme lipoprotein lipase, but the resultant triacylglycerol-depleted remnant particle remained in the plasma instead of being rapidly taken up by the liver. Therefore, the presence of emulsion cholesterol is a critical determinant of early metabolic events, and the findings point to a similar role for cholesterol in the natural triacylglycerol-rich lipoproteins.     

Isolation of serum chylomicrons prior to density gradient ultracentrifugation of other serum lipoprotein classes

A method for the removal of serum chylomicrons before density gradient ultracentrifugation of the other serum lipoproteins using an SW 41 swinging bucket rotor is presented. In a preliminary spin, the chylomicrons with an Sf greater than 400 X 10(-13) s float to the top of the gradient, whereas the other lipoproteins are retained in the infranatant fraction. After removal of the chylomicrons, the other serum lipoproteins are subsequently fractionated by isopycnic density gradient ultracentrifugation. Analysis of the separated lipoprotein fractions suggested that this procedure permits isolation of a chylomicron fraction consisting solely of chylomicrons but that the very low density lipoprotein fraction subsequently isolated also contains chylomicrons or chylomicron remnants with an Sf less than 400 X 10(-13) s, and that there is considerable overlap in flotation rate and particle size of very low density lipoproteins and chylomicrons.     

Effects of cholesterol content on the metabolism of protein-free emulsion models of lipoproteins

After intravenous injection, emulsions with compositions similar to chylomicrons behaved metabolically as described for chylomicrons, with faster removals of triacylglycerols than cholesteryl esters from the blood after injection into rats, and with greater uptakes of cholesteryl esters than triacylglycerols by the liver. In contrast, emulsions with a high content of free cholesterol showed equal removal rates from the blood of triacylglycerols and cholesteryl esters; and similar uptakes by the liver. This pattern of metabolism was that expected for a chylomicron core remnant particle. Emulsions poor in cholesteryl ester but rich in free cholesterol showed remnant-like behavior, whereas emulsions rich in cholesteryl ester but poor in free cholesterol were metabolized like nascent chylomicron particles. The amount of free cholesterol appeared to regulate metabolism by affecting the binding of apolipoproteins to the particle surface. Emulsions with a high content of free cholesterol bound less A-I, A-IV and C apolipoproteins, and the relative amount of apolipoprotein E was increased. All of these effects are consistent with the metabolic differences between chylomicrons and remnant particles, suggesting that the amount of free cholesterol plays a regulatory role in chylomicron metabolism.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.