Cholesterol Acyltransferase Reaction Research Articles

The effect of progesterone on the regulatory mechanisms of biliary cholesterol secretion in the rat.

We tested the hypothesis that progesterone, an inhibitor of cholesterol esterification in liver microsomes, increases biliary cholesterol output by increasing the availability of cholesterol. Initial bile samples of 20 min were obtained from acute bile fistula rats after seven daily doses of progesterone (5 to 55 mg per kg of body weight). Biliary cholesterol output correlated with the doses of progesterone, r = 0.64 (p less than 0.005). A 100% increment in biliary cholesterol output was obtained with progesterone doses of 30 to 55 mg per kg of body weight. Under these conditions, biliary phospholipid output increased 50% (p less than 0.02), but bile salt output remained normal. The relationship between biliary cholesterol and phospholipids as a function of bile salt output was studied after acute depletion of the bile salt pool. A rectangular hyperbola was the best curve fitting for the experimental data in control and progesterone-injected rats. In the physiological range of bile salt output, between 60 and 120 nmoles per gm per min, progesterone-injected rats secreted 100% more biliary cholesterol than did controls. The calculated theoretical maximal cholesterol and phospholipid outputs were significantly increased in progesterone-injected animals. Serum and hepatic cholesterol pool, free and ester fractions, remained normal. The acyl-CoA: cholesterol acyltransferase reaction was 30% inhibited in hepatic microsomes of progesterone-injected rats, (p less than 0.05). The changes in biliary phospholipids and cholesterol output produced by progesterone were rapidly reversed by either 0.5% cholesterol feeding or 2 mg per kg of body weight ethynyl estradiol injection. These manipulations simultaneously produced a 100% increment in the microsomal acyl-CoA: cholesterol acyltransferase activity (p less than 0.005) and increased 4-fold the concentration of hepatic cholesterol esters. This experimental model suggests a functional interrelationship between biliary cholesterol output and the rate at which the liver esterifies cholesterol.

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

Ability of Serum to Decrease Cellular AcylCoA:Cholesterol Acyl Transferase Activity Predicts Cardiovascular Outcomes

We evaluated whether cholesterol efflux activity of serum is associated with the presence of angiographic coronary artery disease (CAD) and the risk of major adverse cardiovascular events (MACE) and death. We studied 168 men undergoing coronary angiography. Cholesterol efflux activity was measured in vitro by incubation of patient serum with human skin fibroblasts and defined as the ability of serum to decrease the pool of cholesterol available for esterification by the acylCoA:cholesterol acyl transferase (ACAT) reaction. We evaluated whether this activity was associated with the presence of CAD and the risk of MACE and death during a 4.5-year follow-up. Serum-induced changes in ACAT activity did not correlate with HDL levels or the presence of CAD. Patients in the highest tertile of change in ACAT activity had a significantly higher risk for MACE (HR, 2.15; 95% CI, 1.36 to 3.39; P=0.001) and death (HR, 2.23; 95% CI, 1.17 to 4.26; P=0.01). These correlations were independent of other risk markers including LDL, HDL, and C-reactive protein levels. Serum-induced depletion of cellular cholesterol available for esterification by ACAT was a strong, independent predictor of MACE and death. We speculate that the ability of serum to decrease ACAT activity depends on ATP binding cassette transporter A1 (ABCA1)-mediated efflux. Furthermore, serum samples that induce larger changes in ACAT activity contain increased levels of HDL particles that preferentially interact with ABCA1 and that these particles accumulate in the serum of patients because of low activity of ABCA1 in vivo preventing or limiting the extent of apoA-I lipidation.

Molecular aspects of intracellular sterol esterification: the acyl coenzyme A: cholesterol acyltransferase reaction.

The conversion of cholesterol to an esterified storage form by the enzyme acyl-coenzyme A: cholesterol acyltransferase, is a critical component of sterol and membrane homeostasis and represents a significant step in atherogenesis. The isolation in 1993 of the first molecular probe of acyl-coenzyme A: cholesterol acyltransferase provided a key to understanding this reaction and its regulation. The sequence is apparently ubiquitous in eukaryotes, often with multiple sequence homologs within an organism. Presented here is a review of the known molecular events that lead to intracellular sterol esterification.

Molecular species of cholesteryl esters formed via plasma lecithin: Cholesterol acyltransferase in fish oil supplemented dogs

Effects of a dietary supplement containing n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), on molecular species of cholesteryl ester (CE) formed via the plasma lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) reaction was studied. A commercially available basal diet was initially fed to 18 normal adult dogs for 10 days prior to supplementing the dogs, divided into two groups of nine, using either menhaden fish (MHO) or safflower (SAF) oil capsules for a 22 day period. Fatty acid compositions of plasma phospholipid (PL) and CE were measured at days 0, 10 and 32. Characterization of the plasma LCAT-derived reaction products formed in vitro was performed using a radiolabeled cholesterol substrate and molecular species formed were separated by argentation thin-layer chromatography. Significant differences in PL EPA, DHA, and docosapentaenoic acid (DPA) were found in the MHO group. The CE fatty acid distributions revealed a greater than 20-fold elevation in EPA and a nearly 6-fold increase in DHA after MHO supplements. However, DPA was not detected in any CE fatty acid samples suggesting that this fatty acid is not a substrate for cholesterol esterification. The CE molecular species in plasma and those formed via the plasma LCAT reaction in vitro support the possibility that LCAT is responsible, at least in part, for the plasma CE fatty acid distribution. Ratios of selected plasma CE and LCAT derived CE n-6 and n-3 fatty acids using the MHO group data resulted in 3-fold elevations of both DHA/EPA and DHA/AA in the LCAT-derived in vitro CE fraction compared to CE in whole plasma. It is concluded that while both EPA and DHA are suitable substrates for CE formation, relatively lower amounts of plasma CE-DHA compared to the LCAT derived CE-DHA indicates that this fatty acid may be selectively taken up by the tissues compared to CE-EPA and AA.

Long-chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein apolipoprotein A-I and the activation energy of the lecithin:cholesterol acyltransferase reaction

The lecithin:cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), [3H]cholesterol, and apo A-I. The PC component of the rHDL contained sn-1 16:0 and sn-2 18:1 (POPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC) or 10% of the respective PC species and 90% sn-1 18:1, sn-2 16:0 PC ether (OPPC ether). The appVmax of the rHDL containing 100% PC varied 10-fold and was ordered POPC > PEPC > PAPC > PDPC, whereas the appKm values varied 19-43 microns PC. The ether-containing rHDL had appVmax values 17-40% of their respective 100% PC rHDL, but maintained the same rank order. The activation energy of LCAT was lower for rHDL containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.2-16 M) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M) and there was a strong correlation (r = 0.71) between LCAT activation energy and the stability of apoA-I (i.e., D1/2). We conclude that long chain PUFA in the sn-2 position of PC decreases the catalytic efficiency of LCAT, the activation energy of the LCAT reaction and the stability of apoA-I on the rHDL particles. The strong association between rHDL apoA-I stability and LCAT activation energy suggests that the temperature-dependent step of the LCAT reaction may be sensitive to the strength of the interaction of apoA-I with rHDL PC.

Effect of lipoprotein-X on lipid metabolism in rat kidney.

Lipoprotein-X (Lp-X) is found in the plasma of patients with familial lecithin: cholesterol acyltransferase (LCAT) deficiency syndromes. The majority of the patients with this disorder develop progressive glomerulosclerosis. In this study, the effect of Lp-X on lipid metabolism in perfused rat kidney was investigated. Lp-X was isolated from plasma of patients with familial LCAT deficiency by sequential ultracentrifugation and gel filtration column chromatography. Rat kidneys were perfused for 1-2 h with Krebs-Henseleit buffer containing 20 microM [1-(14)C]acetate or 20 microM [Me-3H]choline. In the presence of Lp-X, no significant difference in the incorporation of radioactivity into triglycerides, cholesterol, phosphocholine, CDP-choline and sphingomyelin was observed. However, incorporation of radioactivity into cholesteryl esters and phosphatidylcholine was significantly elevated in Lp-X perfused kidneys. The contents of cholesterol, cholesteryl esters and phosphatidylcholine were also significantly increased in Lp-X perfused kidneys. The increase in lipid content in the Lp-X perfused kidney is attributed to the direct deposition of Lp-X lipids into the organ. The increase in the labelling of cholesteryl esters was attributed to the increase of available substrate (cholesterol) for the acyl-CoA:cholesterol acyltransferase (ACAT) reaction. The increase in phosphatidylcholine labelling was caused by a reduced turnover of the newly synthesized labelled phosphatidylcholine during Lp-X perfusion.

Regulation of Cellular Cholesterol Efflux by Lecithin:Cholesterol Acyltransferase Reaction through Nonspecific Lipid Exchange

Erythrocyte was found lacking in reactivity to lipid-free apolipoproteins to generate pre-beta-high density lipoprotein (HDL) with the cellular lipid and, therefore, was used to study cellular cholesterol efflux to plasma lipoproteins exclusively by a nonspecific exchange mechanism. Over the range of hematocrit from 1-20% (cellular cholesterol pool of 2.5 micrograms per 250 microliters), the fractional rate of cellular cholesterol efflux to lipoprotein was constant, and, therefore, absolute efflux rate was a linear function of the hematocrit of this range. In the absence of lecithin:cholesterol acyltransferase (LCAT), the cholesterol influx rate from lipoproteins was equal to the efflux rate from erythrocyte resulting in no net transfer of cholesterol, with either HDL or low density lipoprotein. In the presence of LCAT in the mixture of HDL and erythrocyte, cholesterol was esterified exclusively in HDL regardless of the origin. When the hematocrit was low and efflux of cellular cholesterol was slower than cholesterol esterification, the esterification of cell-originating cholesterol did not directly enhance the efflux. With high hematocrit that gives faster cholesterol efflux, the efflux was increased directly by the cholesterol esterification. On the other hand, the LCAT reaction significantly reduced HDL-cholesterol influx. The LCAT reaction thus induces substantial net cholesterol efflux from erythrocytes through a nonspecific cholesterol exchange mechanism.

Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.

We attempted to demonstrate selective modulation of lipid-free apolipoprotein-mediated cellular lipid efflux in order to test the hypothesis that it is an event independent of nonspecific physicochemical cholesterol exchange. Probucol, a unique cholesterol-lowering drug, was found to selectively suppress this pathway in vitro in mouse peritoneal macrophage. Probucol was given to the cells via the uptake of acetylated low-density lipoprotein (LDL) into which it had been incorporated. The uptake of lipoprotein-cholesteryl ester by the macrophage was the same whether the acetylated LDL was probucol-carrying or probucol-free, and probucol accumulated in the cell in parallel to cholesterol when carried by the lipoprotein. Incorporation of [35S]methionine into cell protein was unaffected by probucol accumulated in the cells. The efflux of cellular cholesterol and phospholipid mediated by lipid-free human apolipoproteins (apo) A-I, A-II, and E was all completely inhibited by probucol. Reversible binding of free apoA-I to the cellular surface was also completely blocked by probucol in this condition. On the other hand, nonspecific cholesterol exchange between LDL and macrophage was unaffected by probucol. Thus, probucol selectively inhibited apolipoprotein-mediated cellular lipid efflux by blocking specific binding of free apolipoprotein to the cell without influencing nonspecific lipid exchange. In the absence of lecithin: cholesterol acyltransferase (LCAT) reaction, apparent cellular cholesterol efflux to high-density lipoprotein (HDL) was reduced by probucol by 40-70% while the rate of cholesterol influx from HDL to the cells was unaffected, resulting in cancellation of the net cellular cholesterol efflux to HDL. However, the increase of the net cholesterol efflux to HDL by LCAT was unaffected by probucol. Net cellular cholesterol efflux to HDL in the absence of LCAT, therefore, seems to depend on an apolipoprotein-mediated mechanism.

Cholesterol Transfer from Low Density Lipoproteins to Reconstituted High Density Lipoproteins Is Determined by the Properties and Concentrations of Both Particles

Cholesterol spontaneously transfers from low density lipoproteins (LDL) to high density lipoproteins (HDL). This transfer is important physiologically as it supplies the major portion of cholesterol for the lecithin:cholesterol acyltransferase reaction and is one mechanism for the reduction of atherogenic LDL cholesterol. The objective of this work was to examine the properties of both HDL and LDL which modulate cholesterol transfer, as well as to obtain the relevant kinetic constants for the transfer at concentrations of lipoproteins approaching those existing in vivo. To examine the effects of HDL structural parameters on cholesterol transfer, we prepared reconstituted HDL particles with saturated or unsaturated phospholipid, with apolipoprotein AI or apolipoprotein AII, with increasing size and phospholipid content, and with increasing initial contents of cholesterol. We also prepared five LDL subfractions of variable density and size. The kinetics of cholesterol mass transfer were measured by incubating LDL with rHDL at 37 degrees C, separating the lipoproteins by dextran sulfate/Mg2+ precipitation of LDL at timed intervals, and analyzing rHDL cholesterol content. The cholesterol content of rHDL at equilibrium, Ceq, and the half-time for transfer, t1/2, as well as the ratio of the lipid surface areas of LDL to rHDL were used in the analysis of the kinetic data by the aqueous diffusion model for lipid transfer developed by Nichols and Pagano [(1982) Biochemistry 21, 1720-1726]. The only variables that significantly affect the C(eq) and/or t1/2 are the phospholipid content and composition of the rHDL and the size or density of the LDL particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Lecithin:cholesterol acyltransferase reaction on cellular lipid released by free apolipoprotein-mediated efflux.

Lecithin:cholesterol acyltransferase (LCAT) reaction was studied in free apolipoprotein-mediated cellular lipid efflux from mouse peritoneal macrophages and human skin fibroblasts. When the cells were incubated with lipid-free human apolipoproteins (apo) A-I or A-II, pre-beta high density lipoprotein (HDL) particles were generated by removing cellular cholesterol and phospholipid. Cholesterol was esterified by LCAT in such particles generated with human apoA-I, but not in those with apoA-II. The reactivity of the apoA-I-pre-beta-HDL particles with LCAT was in the same order as that in human plasma HDL and in phosphatidylcholine/cholesterol unilamellar vesicles activated by apoA-I when compared on the rate of percent cholesterol esterification. However, cholesterol efflux mediated by apoA-I was not enhanced by active cholesterol esterification in the medium from either type of cells. Thus, it is unlikely the LCAT reaction on newly generated pre-beta-HDL directly causes further cellular cholesterol efflux. In control experiments, LCAT esterified cholesterol on human plasma HDL in the cell medium regardless of its origin, either HDL or cells. Cholesterol esterification on HDL was unable to enhance cellular cholesterol efflux significantly but reduced the influx of cholesterol from HDL to cell, resulting in the increase of net efflux of cellular cholesterol, in agreement with the results previously demonstrated.

Lipoprotein phospholipid composition and LCAT activity in nephrotic and analbuminemic rats

Albumin is an acceptor of lysophosphatidylcholine (LPC), product of the lecithin:cholesterol acyl transferase (LCAT) reaction, and it has been suggested that low LCAT activity and reduced cholesterol esterification rate in patients with the nephrotic syndrome may be linked to depletion of albumin. Effects of low plasma albumin levels on LCAT activity, cholesterol esterification rates and LPC-binding were therefore studied in hyperlipidemic nephrotic (NS) and analbuminemic (NAR) rats. LPC-binding was also measured in normoalbuminemic rats with dietary hypercholesterolemia. Remarkably, LCAT activity, measured with excess exogenous substrate, was not decreased but increased in both NAR and NS rats. Molar esterification rates with endogenous substrate were increased in NAR but normal in NS rats. In normoalbuminemic rats, with or without hypercholesterolemia, LPC was primarily found in the lipoprotein-deficient plasma and the HDL3 fraction. In NAR and NS rats LPC levels were increased in lipoproteins (notably in LDL and HDL2), but, in marked contrast to normoalbuminemic rats, decreased in lipoprotein-deficient plasma. Phosphatidylcholine, quantitatively the major phospholipid, was distributed proportionally over the lipoproteins in NS, NAR and control rats. Therefore, in hypoalbuminemia and analbuminemia LPC is mainly bound to lipoproteins, which is in contrast to the paucity of LPC in these particles in normoalbuminemic rats. Cholesterol esterification in nephrotic plasma is thus not impaired by lack of an acceptor for LPC-binding. The absence of an increase in molar cholesterol esterification in conjunction with increased LCAT activity points to a possible defect of the substrate for this reaction in nephrotic plasma. Increased LPC levels in LDL, a characteristic of oxidized LDL, may be a hitherto unrecognized atherosclerotic risk factor in the nephrotic syndrome.

Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3β-[2-(diethylamino) ethoxy]androst-5-en-17-one

The compound U18666A (3β-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to inhibit the cellular transfer of low-density lipoprotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from plasma membranes to intracellular membranes. Translocation of plasma membrane cholesterol was induced by degradation of plasma membrane sphingomyelin. The sphingomyelinase-induced activation of the acyl-CoA cholesterol acyl transferase (ACAT) reaction was completely inhibited in a dose-dependent manner by U18666A, both in cultured human skin fibroblasts and baby hamster kidney cells. Half-maximal inhibition (within 60 min) was obtained with 0.5–1 μg/ml of U18666A. A time-course study indicated that the onset of inhibition was rapid (within 10–15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma membrane cholesterol translocation in sphingomyelinase-treated HSF cells was significantly lowered in the presence of U18666A (at 3 μg/ml). The effect of U18666A on cholesterol translocation was also fully reversible when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [ 3H]cholesterol, the compound U18666A inhibited in a dose-dependent manner the cyclic AMP-stimulated secretion of [ 3H]steroid hormones. The effects seen with compound U18666A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol translocation or ACAT activation. Since different cell types display sensitivity to U18666A in various intracellular cholesterol transfer processes, they appear to have a common U18666A-sensitive regulatory mechanism.

Oxidative tyrosylation of high density lipoprotein by peroxidase enhances cholesterol removal from cultured fibroblasts and macrophage foam cells.

Lipoprotein oxidation is thought to play a pivotal role in atherogenesis, yet the underlying reaction mechanisms remain poorly understood. We have explored the possibility that high density lipoprotein (HDL) might be oxidized by peroxidase-generated tyrosyl radical. Exposure of HDL to L-tyrosine, H2O2, and horseradish peroxidase crosslinked its apolipoproteins and strikingly increased protein-associated fluorescence. The reaction required L-tyrosine but was independent of free metal ions; it was blocked by either catalase or the heme poison aminotriazole. Dityrosine and other tyrosine oxidation products were detected in the apolipoproteins of HDL modified by the peroxidase/L-tyrosine/H2O2 system, implicating tyrosyl radical in the reaction pathway. Further evidence suggests that tyrosylated HDL removes cholesterol from cultured cells more effectively than does HDL. Tyrosylated HDL was more potent than HDL at inhibiting cholesterol esterification by the acyl-CoA:cholesterol acyltransferase reaction, stimulating the incorporation of [14C]acetate into [14C]cholesterol, and depleting cholesteryl ester stores in human skin fibroblasts. Moreover, exposure of mouse macrophage foam cells to tyrosylated HDL markedly diminished cholesteryl ester and free cholesterol mass. We have recently found that myeloperoxidase, a heme protein secreted by activated phagocytes, can also convert L-tyrosine to o,o'-dityrosine. This raises the possibility that myeloperoxidase-generated tyrosyl radical may modify HDL, enabling the lipoprotein to protect the artery wall against pathological cholesterol accumulation.

Structural properties of high density lipoprotein subclasses homogeneous in protein composition and size.

We isolated native high density lipoprotein (HDL) subclasses homogeneous in size and in their protein content with the objective of investigating the differences and similarities in their apolipoprotein AI (apoA-I) structures. Defined particles were isolated from ultracentrifugally prepared HDL by immunoaffinity and gel-filtration chromatography. The isolated 88-A LpAI, 106-A LpAI, 96-A LpAI/AII particles (LpAI, particles contain only apoA-I; LpAI/AII, particles contain apoA-I and apoA-II), together with a 93-A reconstituted HDL were analyzed for purity, composition, and content of apolipoprotein molecules per particle, and were examined by far and near circular dichroism and intrinsic fluorescence spectroscopic methods, as well as by reaction kinetics with lecithin:cholesterol acyltransferase. The spectroscopic analyses indicated that the secondary structures and three-dimensional arrangements of apoA-I in all these particles are remarkably similar: their tryptophan residues are located in similar nonpolar environments and become exposed to increasing concentrations of guanidine hydrochloride in comparable denaturation steps; the 60-65% alpha-helical structures in apoA-I are denatured in similar patterns with 0-5 M denaturant concentrations. However, increasing surface lipid contents and the presence of apoA-II stabilize apoA-I on the HDL particles. The reaction kinetics with lecithin:cholesterol acyltransferase are similar and slow for the isolated HDL particles, reflecting product inhibition, and/or an apoA-I conformation that is unfavorable for the activation of the lecithin:cholesterol acyltransferase reaction.

Hepatic ACAT activity in African green monkeys is highly correlated to plasma LDL cholesteryl ester enrichment and coronary artery atherosclerosis.

Previous studies and this study of African green monkeys show a strong positive correlation between plasma low density lipoprotein (LDL) size and the extent of coronary artery atherosclerosis (CAA). Increased LDL size was principally due to the accumulation of cholesteryl oleate molecules within the particle core, suggesting that many of these cholesteryl esters were of tissue origin, i.e., from the acyl-coenzyme A:cholesterol acyltransferase (ACAT) reaction instead of the lecithin:cholesterol acyl-transferase (LCAT) reaction. The current study was conducted to test the hypothesis that ACAT in the liver is the source of the increased numbers of cholesteryl oleate molecules in plasma LDL particles that appear to increase the atherogenic potential of LDL. Monkeys were fed diets rich in fat (lard, safflower oil, or fish oil) and cholesterol for 3-6 years before liver perfusion, ACAT assay, and evaluation of CAA. Hepatic ACAT activity was positively correlated with hepatic cholesteryl ester secretion (r = 0.61, p < 0.001), plasma LDL cholesteryl ester content (r = 0.60, p < 0.0001), and the extent of CAA (r = 0.62, p < 0.0001). The number of cholesteryl oleate molecules within LDL increased proportionally with LDL size in each of the diet groups. Hepatic cholesteryl oleate concentration was correlated with the accumulation of cholesteryl oleate in liver perfusate (r = 0.72, p < 0.01) and with plasma LDL cholesterol oleate content (r = 0.73, p < 0.0001). Our interpretation is that these data, obtained in a relevant primate model of CAA, suggest that hepatic ACAT increases the atherogenicity of LDL by augmenting both the secretion by the liver and accumulation in plasma LDL of cholesteryl oleate.

The effect of borage oil consumption on human plasma lipid levels and the phosphatidylcholine and cholesterol ester composition of high density lipoprotein

The effect of consuming an enriched source of gamma-linolenic acid (GLA, 18:3 n-6) in the form of borage oil on the plasma lipid levels of human volunteers and the fatty acid composition of high density lipoprotein (HDL)-phosphatidylcholine (PC) and HDL-cholesterol ester (CE) was examined. Furthermore, an estimation was made of the relative utilization of GLA and dihomo-gamma-linolenic acid (DGLA, 20:3 n-6) by the plasma lecithin: cholesterol acyltransferase (LCAT) reaction. For this purpose, six healthy male subjects consumed encapsulated borage oil (21.8 wt% GLA) so as to consecutive days followed by a wash-out period of an additional 42 days. Analysis of plasma lipids (triglycerides, total cholesterol, HDL-cholesterol, low density lipoprotein (LDL)-cholesterol, and the plasma HDL-cholesterol:total cholesterol ratio indicated no significant change in any of the latter arising from the supplementation. Consumption of the borage oil supplement was found to produce marked alterations in the fatty acid compositions of HDL-PC and HDL-CE. Whereas only a moderate accumulation in PC (up to 0.6 mol %) of GLA was found with supplementation, a more dramatic rise in DGLA (by 3.9 mol %) and arachidonic acid (AA, 20:4 n-6) (by 3.3 mol%) was found. These changes were reversible by day 64. In contrast to the HDL-PC, the GLA accumulation in HDL-CE exceeded that of DGLA such that the net rise was 1.8 mol % as compared to only 0.8 mol % for DGLA. Positional analysis of the HDL-PC from subjects at day 43 revealed GLA and DGLA to reside almost exclusively in the 2-position (>98%). Dividing the percentages of unsaturated fatty acids in the HDL-CE relative to their levels in the corresponding 2-position of PC provided the following estimated order of fatty acid selectivity for the plasma LCAT reaction: GLA> linoleic acid (LA, 18:2 n-6)> oleic acid (OA, 18:1 n-9)>AA>DGLA≥docosahexaenoic acid (DHA, 22:6 n-3).

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of hypoxanthine and other purines on the lecithin: cholesterol acyltransferase reaction in the plasma of rat and other species

1. 1. Esterification of radiolabelled cholesterol in the plasma of rat, mouse, pig, ox and, to a lesser extent, guinea pig was partially inhibited by hypoxanthine, xanthine and guanine; esterification in human plasma and in plasma from 12 other vertebrate species was unaffected by purines. 2. 2. Esterification of endogenous cholesterol and the formation of lysolecithin in rat plasma were decreased in the presence of purines indicating that it was the lecithin: cholesterol acyltransferase (LCAT) reaction that was inhibited rather than the isotopic equilibration of labeled cholesterol with the endogenous substrate lipoproteins 3. 3. Maximum inhibition of the LCAT reaction in rat plasma occurred at 1.4 mM hypoxanthine or xanthine; inhibition was not dependent upon the concentration of LCAT or plasma lipoproteins but increased with the amount of lipoprotein depleted rat plasma (LDRP) present in the incubation mixture. 4. 4. Partial inhibition of the LCAT reaction in rat or mouse plasma by purines had no significant effect on the fatty acyl composition of the cholesteryl esters (CE) formed by LCAT. 5. 5. In the presence of heated rat plasma, LDRP or, to a lesser extent, rat high density lipoproteins (HDL) prepared from heated plasma, the LCAT reaction in human plasma was inhibited by hypoxanthine. 6. 6. Rat HDL and LDRP prepared from plasma pre-incubated at 37°C for 4 hr before heating increased and decreased, respectively, the inhibitory effect of hypoxanthine on human plasma LCAT compared with HDL and LDRP prepared from unincubated rat plasma. 7. 7. These findings suggest that purines inhibited the LCAT reaction in rat plasma not through any direct effect on the enzyme, but by interacting with a protein present both in HDL and in LDRP; this protein may be an apolipoprotein co-factor of LCAT not found in human plasma.