Discoidal High-density Lipoprotein Particles Research Articles

Lecithin-cholesterol acyltransferase. Assay of cholesterol esterification and phospholipase A2 activities.

AbstractLecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43) is a 67 kDa glycoprotein that is responsible for cholesterol esterification in plasma (1). LCAT has an important physiological role in the maturation of nascent, discoidal high density lipoproteins (HDL) to mature, spherical HDL, through the generation of a cholesteryl ester (CE)-enriched core (2,3). The enzyme has also been implicated in the reverse cholesterol transport pathway, which results in the net transport of cholesterol from peripheral tissues back to the liver for excretion (1,4). The central role of LCAT in these physiological processes is supported by the finding of plasma and tissue accumulation of free cholesterol and the presence of nascent, discoidal HDL particles in the plasma of familial LCAT-deficient subjects (5).KeywordsCholesteryl EsterCholesterol EsterificationHigh Density LipoproteinFree CholesterolShake Water BathThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Imaging and manipulation of high-density lipoproteins

The atomic force microscope (AFM) has been used to image a variety of biological systems, but has rarely been applied to soluble protein-lipid complexes. One of the primary physiological protein-lipid complexes is the high-density lipoproteins (HDL), responsible for the transport of cholesterol from the peripheral tissues and other lipoproteins to the liver. We have used the AFM to directly image discoidal reconstituted HDL (rHDL) particles for the first time. The height of these particles is consistent with a phospholipid bilayer structure, but careful high resolution measurements of particle diameters has indicated that they fuse when adsorbed to mica. Furthermore, it has been demonstrated that the AFM can be used to initiate this bilayer fusion in a controlled manner, allowing the fabrication of stabilized, nanometer scale, phospholipid bilayer "domains."

Modifications in High-Density Lipoprotein Lipid Composition and Structure Alter the Plasma Distribution of Free and Liposomal Annamycin

Recent studies have shown that changes in lipoprotein cholesterol and triglyceride concentration alters the plasma distribution of free (Ann.) and liposomal annamycin (LAnn) and that the majority of Ann. is associated with high-density lipoproteins (HDL) following the incubation in plasma of LAnn. To demonstrate that alterations in HDL lipid composition and HDL structure may influence the plasma distribution of Ann. and LAnn, Ann. and LAnn (20 ,μg/mL) were incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits the conversion of free cholesterol to esterified cholesterol) 18h prior to the experiment or in untreated plasma for 60min at 37 °C. In addition, Ann. and LAnn were co-incubated with DTNB in plasma for 6min at 37 °C. Following incubation the plasma was separated into its HDL, low- density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for Ann. by fluorimetry. The HDL plasma cholesterol:triglyceride concentration ratio was significantly decreased following 18h of DTNB pretreatment compared to untreated plasma controls. No significant differences in LDL/VlDL plasma cholesterol:triglyceride concentration ratio following 18h of DTNB pretreatment was observed. An increased number of discoidal HDL particles were observed following 18h of DTNB pretreatment. When Ann. was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL, LDL, and VLDL fractions significantly increased. However, the percentage of Ann. recovered within the LPDP fraction was significantly decreased. When LAnn was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL fraction significantly decreased. The percentage of Ann. recovered in the LPDP fraction significantly increased when LAnn was incubated in plasma pretreated with DTNB for 18 h. No significant differences in Ann. lipoprotein distribution were observed when Ann. and LAnn were co-incubated with DTNB in plasma for 1 h. These findings suggest that the cholesterol:triglyceride concentration ratio and physical structure of HDL maybe important in defining the capacity of hDl to sequester Ann.

Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL

The effects of lipid transfers and hepatic lipase (HL) on the concentration, composition, particle size distribution and morphology of high density lipoproteins (HDL) have been investigated. Human plasma supplemented with additional very low density lipoproteins (VLDL), cholesteryl ester transfer protein (CETP) and HL has been incubated at 37°C for up to 8 h. The HDL became depleted of cholesteryl esters and reduced in particle size. Within 2 h of such incubation they had also lost about 30% of their apo A-I. However, with extension of the incubations beyond 2 h, the apo A-I returned progressively to the HDL fraction until, after 8 h, the concentration of apo A-I in HDL was identical to that in non-incubated samples. This return of apo A-I to the HDL density range was accompanied by a progressive appearance in electron micrographs of discoidal HDL particles. Thus, the depletion of the core lipid content and the reduction in particle size of HDL promoted by lipid transfers and HL activity in vitro is accompanied by a shedding of apo A-I which forms the nucleus of new discoidal HDL particles. The potential physiological importance of such a process is considerable.

Lecithin:cholesterol acyltransferase-induced modifications of liver perfusate discoidal high density lipoproteins from African green monkeys.

The role of lecithin:cholesterol acyltransferase (LCAT) in the formation of plasma high density lipoproteins (HDL) was studied in a series of in vitro incubations in which perfusates from isolated African green monkey livers were incubated at 37 degrees C with partially purified LCAT for between 1 and 13 hr. The HDL particles isolated from monkey liver perfusate stored at 4 degrees C and not exposed to added LCAT contained apoA-I and apoE, were deficient in neutral lipids, and were observed by electron microscopy as discoidal particles. Particle sizes, measured as Stokes' diameters by gradient gel electrophoresis (GGE), ranged between 7.8 nm and 15.0 nm. The properties of perfusate HDL were unchanged following incubation at 37 degrees C in the presence of an LCAT inhibitor. However, HDL subfractions derived from incubations at 37 degrees C with active LCAT contained apoA-I as the major apoprotein, appeared round by electron microscopy, and possessed chemical compositions similar to plasma HDL. The HDL isolated from perfusate incubations at 37 degrees C with low amounts of LCAT had a particle size and chemical composition similar to plasma HDL3a. In three of four perfusates incubated with higher levels of LCAT activity, the HDL products consisted of two distinct HDL subpopulations when examined by GGE. The major subpopulation was similar in size and composition to plasma HDL2a, while the minor subpopulation demonstrated the characteristics of plasma HDL2b. The data indicate that the discoidal HDL particles secreted by perfused monkey livers can serve as precursors to three of the major HDL subpopulations observed in plasma.

32] Reconstitution of high-density lipoproteins

The detailed investigation at the molecular level of protein–lipid interactions, functions, and metabolism of high-density lipoproteins (HDL) has led to the development of several reconstitution methods for micellar particles of defined chemical composition, having the overall size and density of HDL. This chapter describes two approaches for the codispersion and interaction of apolipoproteins with various lipids: spontaneous reaction of vesicles with apolipoproteins and the detergent-mediated reconstitution methods. Both of these methods give discoidal particles with apolipoproteins, phosphatidylcholines (PC), and free cholesterol of dimensions and composition analogous to nascent, discoidal HDL particles. Although spontaneous reaction of PC vesicles with apolipoproteins is kinetically controlled by the physical state and composition of the lipid vesicles, and is restricted to only a few PC types; the detergent reconstitution method is based on the dispersion of lipids in sodium cholate, which allows the incorporation of a much larger variety of phospholipids into discoidal, HDL-like particles. Thus, the synthetic discoidal complexes can be used as chemically and physically defined structural and functional models of nascent HDL with confidence.

Effect of biliary diversion on rat mesenteric lymph apolipoprotein-I and high density lipoprotein.

The effect of biliary diversion on intestinal apolipoprotein (apoA)-I and high density lipoprotein formation was studied in mesenteric lymph fistula rats. Bile diversion was produced by an exteriorized catheter that allowed interruption and reconstitution of the enterohepatic circulation. Bile diversion reduced lymph cholesterol output from 0.47+/-0.05 mumol/h to 0.17+/-0.03 mumol/h (P < 0.025), and lymph triglyceride output from 3.6+/-0.3mumol/h to 0.6+/-0.05 mumol/h (P < 0.025) after 24 h. This was due to depletion of lymph chylomicrons and very low density lipoprotein (VLDL). Despite the reduced lipid outputs, lymph apoA-I output was maintained during biliary diversion (basal: 119+/-15 mug/h; diverted 140+/-20 mug/h, n = 12). During biliary diversion, high density lipoprotein (HDL) were maintained in mesenteric lymph as shown by lipoprotein and immunoelectrophoresis. Bile diversion altered the lipid composition of lymph HDL. Bile-diverted lymph HDL was depleted in total cholesterol and has a greater phospholipid/cholesterol ester ratio than basal lymph HDL. Lymph HDL contained discoidal particles when examined by negative stain electron microscopy. Bile diversion was associated with a reduction in the size of discoidal HDL particles (basal, nondiverted, 165+/-7A (n = 112) compared with diverted 126+/-5A (n = 98, P < 0.025). Experiments were then carried out to determine the source of the apoA-I and HDL found in lymph from bile-diverted animals. The transfer of HDL from plasma into lymph was determined by the intravenous infusion of (125)I-apoA-I labeled HDL into lymph fistula rats. In both nonbile-diverted and diverted rats, the specific activity of apoA-I in the HDL fraction of lymph was 23% of the specific activity of apoA-I in plasma HDL, indicating that the major portion (75%) of mesenteric lymph apoA-I did not come from plasma filtration. In other experiments the intraduodenal infusion of [(3)H]leucine to bile fistula, lymph fistula rats resulted in relative fivefold increase in the specific activity in apoA-I in lymph HDL when compared with the specific activity of apoA-I in plasma HDL from the same animal. We conclude that intestinal apoA-I secretion is maintained during biliary diversion and that synthesis of this apoprotein occurs in the absence of chylomicron formation. We also conclude that discoidal HDL are present in mesenteric lymph despite reduced triglyceride absorption and secretion into lymph.