Postprandial chylomicrons: potent vehicles for transporting cholesterol from endogenous LDL+HDL and cell membranes to the liver via LCAT and CETP

We examined whether postprandial (PP) chylomicrons (CMs) can serve as vehicles for transporting cholesterol from endogenous cholesterol-rich lipoprotein (LDL+HDL) fractions and cell membranes to the liver via lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities. During incubation of fresh fasting and PP plasma containing [(3)H]cholesteryl ester (CE)-labeled LDL+HDL, both CMs and VLDL served as acceptors of [(3)H]CE or cholesterol from LDL+HDL. The presence of CMs in PP plasma suppressed the ability of VLDL to accept [(3)H]CE from LDL+HDL. In reconstituted plasma containing an equivalent amount of triglycerides from isolated VLDL or CMs, a CM particle was about 40 times more potent than a VLDL particle in accepting [(3)H]CE or cholesterol from LDL+HDLs. When incubated with red blood cells (RBCs) as a source for cell membrane cholesterol, the cholesterol content of CMs, VLDL, LDL, and HDL in PP plasma increased by 485%, 74%, 13%, and 30%, respectively, via LCAT and CETP activities. The presence of CMs in plasma suppressed the ability of endogenous lipoproteins to accept cholesterol from RBCs. Our data suggest that PP CMs may play an important role in promoting reverse cholesterol transport in vivo by serving as the preferred ultimate vehicle for transporting cholesterol released from cell membranes to the liver via LCAT and CETP.     

High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation

The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL(2) and HDL(3), by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.     

Regulation of plasma lecithin:cholesterol acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.

Plasma lecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects. Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL(2), HDL(3), and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes. HDL(2) lipids and protein increased substantially, characterized primarily by enrichment with lecithin. HDL(3), which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others. Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity. The VHD cholesteryl esters and lysolecithin were also reduced. Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism. In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL(3) and VDH, but not in HDL(2), during incubation. Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteins is an unlikely explanation for activation. Since VHD and HDL(3), which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made. The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo. Rose, H. G., and J. Juliano. Regulation of plasma lecithin: cholesteryl acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.     

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.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

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.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

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.     

Effect of growth hormone replacement therapy on plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities in growth hormone-deficient adults

The effects of growth hormone (GH) replacement on plasma lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP), factors involved in high density lipoprotein (HDL) metabolism, are unknown. We carried out a 6 months study in 24 GH-deficient adults who were randomized to placebo (n = 8), low dose GH (1 U daily, n = 8), and high dose GH (2 U daily, n = 8), followed by a 6 months open extension study with high dose GH (1 drop-out). No significant changes in plasma lipoproteins, LCAT, CETP, and PLTP activities, cholesterol esterification (EST) and cholesteryl ester transfer (CET) were observed after placebo. After 6 months of GH (combined data, n = 24), very low + low density lipoprotein (VLDL + LDL) cholesterol (P < 0.05) and apolipoprotein B (P < 0.05) decreased, whereas HDL cholesterol and HDL cholesteryl ester increased (P < 0. 05). Prolonged treatment showed comparable effects. Plasma apolipoprotein A-I and Lp[a] remained unchanged. Plasma LCAT (P < 0. 01) and CETP activities (P < 0.01), as well as EST (P < 0.01) and CET decreased (P < 0.01) after 12 months of GH (n = 15), but PLTP activity did not significantly change. Changes in EST and CET after 12 months of treatment were independently related to changes in plasma LCAT (P = 0.001 and CETP activity (P = 0.01). In conclusion, GH replacement therapy improves the lipoprotein profile in GH-deficient adults. Chronic GH replacement lowers plasma LCAT and CETP activities, contributing to a decrease in cholesterol esterification and cholesteryl ester transfer. These effects may have consequences for HDL metabolism and reverse cholesterol transport.     

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.     

Action of a microbial glycerophospholipid:cholesterol acyltransferase on plasma from normal and LCAT-deficient subjects

The action of a bacterial acyltransferase similar in overall reaction mechanism to the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been studied using normal plasma and lipoproteins and plasma from LCAT-deficient patients. The microbial enzyme (GCAT) catalyzed acyl transfer using phosphatidylcholine and cholesterol in all of the lipoprotein fractions, presumably because it has no apolipoprotein cofactor. In addition, the enzyme was capable of hydrolyzing cholesteryl ester in lipoproteins but not in small unilamellar vesicles nor in micellar dispersions containing low amounts of Triton X-100. This suggests that cholesteryl ester is exposed on the surface of lipoprotein particles or that it may be transferred there quickly from the interior. Although considerable interconversion of radiolabeled cholesterol and cholesteryl ester could be demonstrated upon treatment of normal plasma or lipoproteins with the enzyme, there was little change in the actual amount of either steroid. This indicates that the rate of cholesteryl ester formation is very similar to the rate of hydrolysis. The relative proportions of cholesterol and cholesteryl ester in normal plasma are therefore near the equilibrium ratio for the reaction carried out by GCAT, or the ratio is controlled by the properties of the lipoproteins themselves. During reaction with the microbial acyltransferase, the ratio of cholesterol to cholesteryl ester in plasma from LCAT-deficient patients was reduced substantially, suggesting that the enzyme may have some practical applications.     

The lecithin-cholesterol acyl transferase activity of rat intestinal lymph.

The lecithin-cholesterol acyl transferase (LCAT) activity in rat mesenteric lymph was examined as a possible source of chylomicron cholesteryl ester. Lymph activity was only 2-3% of rat serum activity. Removal of d less than 1.006 lipoproteins increased lymph LCAT activity, but only to 6-8% of that of serum. Relative to total cholesterol in the d greater than 1.08 g/ml fractions, lymph LCAT activity in lymph from fasting rats was less than serum, but in lymph from nonfasting rats the ratio LCAT/HDL-cholesterol reached levels greater than serum, suggesting a contribution of enzyme from the gut. Both LCAT activity and HDL concentration in mesenteric lymph increased during feeding. Subfractions of lymph that inhibited serum LCAT were: chylomicrons, VLDL, chylomicron lipid, VLDL apoprotein, and HDL apoprotein. In the rat, the low LCAT activity of mesenteric lymph was in part due to the low enzyme concentration present, and the activity was apparently lowered further by lipid-rich lipoproteins that inhibited the reaction. Enzyme inhibition due to the apoprotein fractions of lipoproteins is probably minor in the rat in vivo.     

Affinity of lipid transfer protein for lipid and lipoprotein particles as influenced by lecithin-cholesterol acyltransferase

The effects of lecithin-cholesterol acyltransferase (LCAT) on the transfer of cholesterol esters mediated by lipid transfer protein (LTP) and its affinity for lipid and lipoprotein particles were investigated. When the single bilayer vesicle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apolipoprotein- (apo) A-I at the molar ratio of 90:30:1.2:0.18) or high density lipoprotein 3 (HDL3) were used as the cholesteryl ester donor and low density lipoproteins (LDL) as the acceptor, the transfer activity of LTP was enhanced by the addition of low concentrations of LCAT. In contrast, no enhancement of cholesteryl ester transfer was observed upon addition of LCAT to either the discoidal bilayer particle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apo-A-I at the molar ratio of 90:30:1.2:1.0) or high density lipoprotein 2 (HDL2). Although both apo-A-I and apo-A-II promoted the transfer of cholesteryl ester from vesicles to LDL, the additional enhancement of the transfer by LCAT was observed only with the vesicles containing apo-A-I. Gel permeation chromatography of LTP/vesicle and LTP/HDL3 mixtures in the presence and absence of LCAT showed that the affinity of LTP for both the vesicles and HDL3 increased upon addition of LCAT. In contrast, neither HDL2 nor discoidal bilayer particles showed any significant enhancement of LTP binding upon addition of LCAT. By using LCAT covalently bound to Sepharose 4B, a maximal interaction between LTP and bound LCAT was shown to occur at the ionic strength of 0.16. Deviation from this ionic strength reduced the extent of the interaction. At the ionic strength of 0.01 and 0.5, the elution volume of LTP was identical to that of bovine serum albumin.     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

Triacylglycerol increase in plasma very low density lipoproteins in cyclophosphamide-treated rabbit: relationship with cholesteryl ester transfer activity

We have studied the cholesteryl ester transfer between HDL and VLDL in cyclophosphamide-treated rabbits, in order to explain the abnormal cholesteryl ester partition between these two lipoprotein classes. The hypertriglyceridemia caused by treatment with the drug was associated with cholesteryl ester- and triacylglycerol-rich VLDL and with HDL poor in esterified cholesterol but relatively enriched in triacylglycerol. These two lipoprotein classes were characterized by their chemical composition and by gel filtration chromatography. VLDL particles were slightly larger in size, compared with controls. Different transfer combinations were envisaged between these abnormal lipoproteins and control ones. The transfer study involved the plasma fraction of d greater than 1.21 g/ml containing the cholesteryl ester transfer protein (CETP). It appeared that the chemical composition of lipoproteins was responsible for the level of cholesteryl ester transfer between lipoproteins. Actually, when the cholesteryl ester acceptor lipoproteins (VLDL) were enriched in triacylglycerol, the transfer was enhanced. Therefore, the effect of lipolysis on the transfer has also been explored. Lipoprotein lipase seemed to enhance the transfer of cholesteryl ester from HDL to VLDL when these lipoproteins were normal, but an important decline was obtained when triacylglycerol-rich VLDL were lipolyzed. This study defines the relationship between lipoprotein chemical composition and transfer activity of cholesteryl ester from HDL to VLDL.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

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.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.