Probucol normalizes cholesteryl ester transfer in type 2 diabetes.

AIMS: Accelerated cholesteryl ester transfer (CET) protein (CETP) activity is believed to promote macrovascular disease in patients with type 2 diabetes (T2D) by increasing the cholesterol burden of the apoB - containing triglyceride-rich lipoprotein (TGRLP) CE acceptors and promoting small dense LDL formation. While previous studies have shown that this same abnormality is present in patients with type 1 diabetes (T1D) and was normalized by the anti-oxidant drug probucol, its effects on CET in T2D are unknown. PATIENTS AND METHODS: The net mass transfer of CE from HDL to the apoB lipoproteins (VLDL+LDL) was studied in intact plasma from seven T2D patients before and two months after treatment with probucol (1g/day). RESULTS: Before treatment, CET was significantly greater than controls at 1 and 2h (p<.005). Recombination studies showed that this disturbance was attributable to dysfunction of VLDL and not due to altered behavior of HDL or CETP. Probucol treatment normalized CET in all subjects and significantly lowered plasma cholesterol (pre-Rx: 197±4.5 vs post-Rx: 162±27.1mg/dL; mean±S.D.; p<.025) and HDL-C (pre-Rx: 46.4±7.5 vs post-Rx: 39.1±4.0; p<.025) without changing glycemic control. CONCLUSIONS: By normalizing CET in T2D, probucol likely reduces the formation of atherogenic lipoproteins. This effect on CET is achieved through qualitative alterations in CETP's lipoprotein substrates and not through changes in CETP or HDL. Since probucol also has potent anti-oxidative and anti-inflammatory properties, it may have a new role to play in lipoprotein remodeling that reduce cardiovascular risk in T2D.

Molecular dynamic simulation study of cholesterol and conjugated double bonds in lipid bilayers.

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is believed to have anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be associated with anti-obesity effects. In this paper we extend earlier molecular dynamics (MD) simulations of pure CLA-phosphatidylcholine bilayers to investigate the comparative effects of cholesterol on bilayers composed of the two respective isomers. Simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed in which, for each isomer, the simulated bilayers contained 10% and 30% cholesterol (Chol). From MD trajectories we calculate and compare structural properties of the bilayers, including areas per molecule, thickness of bilayers, tilt angle of cholesterols, order parameter profiles, and one and two-dimensional radial distribution function (RDF), as functions of Chol concentration. While the structural effect of cholesterol is approximately the same for both isomers, we find differences at an atomistic level in order parameter profiles and in two-dimensional radial distribution functions.

Conjugated double bonds in lipid bilayers: a molecular dynamics simulation study.

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.

Sphingomyelin: a natural modulator of membrane homeostasis and inflammation.

Although membrane sphingomyelin (SPH) serves as the precursor for many signaling molecules, its presence in large amounts, and its specific localization in the outer monolayer of the plasma membrane suggest that it may have a cytoprotective function. We propose that SPH helps maintain the integrity of the plasma membrane by protecting phosphatidylcholine (PC) against oxidative damage and phospholipase degradation. Since it contains mostly saturated longchain hydrocarbon groups, we postulate that SPH impedes the lateral propagation of the lipid peroxides by decreasing membrane fluidity, while also acting as an 'insulating' molecule. By virtue of its structural similarity to PC, it acts as a competitive inhibitor of the phospholipases, which may otherwise hydrolyze PC excessively. Because phospholipase reaction is the rate-limiting step in eicosanoid synthesis, SPH may serve as an endogenous anti-inflammatory molecule.

Importance of the free sulfhydryl groups of lecithin-cholesterol acyltransferase for its sensitivity to oxidative inactivation.

Lecithin-cholesterol acyltransferase (LCAT) of human plasma is known to be highly susceptible to oxidative inactivation, although the mechanism of this inactivation is unknown. We tested the hypothesis that the high sensitivity of the enzyme is due to the derivatization of its two free SH groups flanking the active site pocket. Modification of the SH groups with a reversible inhibitor protected the enzyme against oxidative inactivation. Mutagenesis of either of the cysteines to glycine increased the resistance of the enzyme, which retained 46% of activity in presence of 150 microM Cu(2+), compared to only 27% of the activity retained by the wild type enzyme (WT). Replacement of both the cysteines with glycines resulted in retention of over 65% activity. Cysteine replacement similarly protected the enzyme from inactivation by the oxidized substrate. Chicken LCAT, which has only one cysteine (Cys(26)), was more resistant than the human enzyme. Introduction of an additional cysteine corresponding to the second cysteine in human LCAT (N184C) resulted in increased susceptibility of chicken enzyme (87% loss of activity in presence of 150 microM Cu(2+), compared to 55% loss in WT). Substitution of the lone cysteine with glycine (C26G) resulted in a more resistant enzyme, which lost <40% activity under the same conditions. These results show that the primary targets of the oxidizing agents or the products of oxidation are the SH groups of the enzyme, whose derivatization leads to steric inhibition of the activity.

Novel physiological function of sphingomyelin in plasma. Inhibition of lipid peroxidation in low density lipoproteins.

Although sphingomyelin (SPH) is a major constituent of all lipoproteins, its physiological function in plasma is not known. In this study, we tested the hypothesis that SPH inhibits lipid peroxidation in low density lipoproteins (LDL) because of its effects on surface fluidity and packing density and that the relative resistance of the buoyant LDL to oxidation, compared with the dense LDL, is partly due to their higher SPH content. Depletion of SPH by treatment with SPHase resulted in shortened lag times and increased rates of oxidation in both LDL subfractions, as measured by the conjugated diene formation in the presence of Cu(2+). Oxidation of LDL by soybean lipoxygenase was similarly stimulated by the degradation of SPH. Oxidation-induced fluorescence decay of diphenylhexatriene-labeled phosphatidylcholine (PC), equilibrated with LDL-PC, was accelerated significantly by the enzymatic depletion of SPH from the lipoprotein. Oxidation of 16:0-18:2 PC in the proteoliposomes was inhibited progressively by the incorporation of increasing amounts of egg SPH into the liposomes. Treatment of SPH-containing proteoliposomes with SPHase reversed the effect of SPH, showing that the presence of intact SPH is necessary for the inhibition of oxidation. Although the incorporation of SPH into the same liposome as the PC (intrinsic SPH) protected the PC against oxidation, the addition of SPH liposomes to PC liposomes (extrinsic SPH) was not effective. Oxidation of 16:0-18:2 PC in liposomes was also inhibited by the incorporation of dipalmitoyl-PC, but not by free cholesterol. These results suggest that SPH acts as a physiological inhibitor of lipoprotein oxidation, possibly by modifying the fluidity of the phospholipid monolayer and thereby inhibiting the lateral propagation of the lipid peroxy radicals.

Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor-acetyl hydrolase-deficient plasma.

To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

All ApoB-containing lipoproteins induce monocyte chemotaxis and adhesion when minimally modified. Modulation of lipoprotein bioactivity by platelet-activating factor acetylhydrolase.

Mildly oxidized LDL has many proinflammatory properties, including the stimulation of monocyte chemotaxis and adhesion, that are important in the development of atherosclerosis. Although ApoB-containing lipoproteins other than LDL may enter the artery wall and undergo oxidation, very little is known regarding their proinflammatory potential. LDL, IDL, VLDL, postprandial remnant particles, and chylomicrons were mildly oxidized by fibroblasts overexpressing 15-lipoxygenase (15-LO) and tested for their ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. When conditioned on 15-LO cells, LDL, IDL, but not VLDL increased monocyte chemotaxis and adhesion approximately 4-fold. Chylomicrons and postprandial remnant particles were also bioactive. Although chylomicrons had a high 18:1/18:2 ratio, similar to that of VLDL, and should presumably be less susceptible to oxidation, they contained (in contrast to VLDL) essentially no platelet-activating factor acetylhydrolase (PAF-AH) activity. Because PAF-AH activity of lipoproteins may be reduced in vivo by oxidation or glycation, LDL, IDL, and VLDL were treated in vitro to reduce PAF-AH activity and then conditioned on 15-lipoxygenase cells. All 3 PAF-AH-depleted lipoproteins, including VLDL, exhibited increased stimulation of monocyte chemotaxis and adhesion. In a similar manner, lipoproteins from Japanese subjects with a deficiency of plasma PAF-AH activity were also markedly more bioactive, and stimulated monocyte adhesion nearly 2-fold compared with lipoproteins from Japanese control subjects with normal plasma PAF-AH. For each lipoprotein, bioactivity resided in the lipid fraction and monocyte adhesion could be blocked by PAF-receptor antagonists. These data suggest that the susceptibility of plasma lipoproteins to develop proinflammatory activity is in part related to their 18:1/18:2 ratio and PAF-AH activity, and that bioactive phospholipids similar to PAF are generated during oxidation of each lipoprotein. Moreover, LDL, IDL, postprandial remnant particles, and chylomicrons and PAF-AH-depleted VLDL all give rise to proinflammatory lipids when mildly oxidized.

Modulation of the positional specificity of lecithin-cholesterol acyltransferase by the acyl group composition of its phosphatidylcholine substrate: role of the sn-1-acyl group.

Human lecithin-cholesterol acyltransferase (LCAT), which is normally specific for the sn-2 position of phosphatidylcholine (PC), derives a significant percentage of acyl groups from the sn-1 position, when sn-2 is occupied by 18:0, 20:4, or 22:6. We investigated the relative importance of the two acyl groups of PC in determining the positional specificity by first analyzing the cholesteryl esters formed in the presence of symmetric PCs labeled at sn-2. Both human and rat LCATs transferred exclusively the sn-2-acyl group from all symmetric PCs, including 18:0-18:0, and 20:4-20:4, showing that the presence of these fatty acids at sn-2 does not automatically alter the positional specificity. The role of the sn-1-acyl group was then tested by using PCs containing 20:4 or 18:0 at sn-2 and fatty acids of various chain lengths and unsaturation at sn-1. With 20:4 at sn-2 and saturated fatty acids of various chain lengths at sn-1, human and rat LCATs derived, respectively, 5-72% and 1-20% of the total acyl groups from the sn-1 position. However, the chain length of the sn-1-acyl did not correlate with its utilization by either enzyme. Various unsaturated fatty acids at sn-1 also were transferred by human LCAT at a higher rate (5-75% of total) than they were transferred by rat LCAT (0-21%). With sn-2-18:0 PCs, however, rat LCAT exhibited greater alteration in positional specificity (30-95% from sn-1) than human LCAT (15-83% from sn-1). These results show that while the primary determinant of positional specificity is the sn-2-acyl group of PC, the structure of sn-1-acyl significantly modifies it.

Trans unsaturated fatty acids inhibit lecithin: cholesterol acyltransferase and alter its positional specificity.

Although dietary trans unsaturated fatty acids (TUFA) are known to decrease plasma HDL, the underlying mechanisms for this effect are unclear. We tested the hypothesis that the decreased HDL is due to an inhibition of lecithin:cholesterol acyltransferase (LCAT), the enzyme essential for the formation of HDL, by determining the activity of purified LCAT in the presence of synthetic phosphatidylcholine (PC) substrates containing TUFA. Both human and rat LCATs exhibited significantly lower activity (-37% to -50%) with PCs containing 18:1t or 18:2t, when compared with the PCs containing corresponding cis isomers. TUFA-containing PCs also inhibited the enzyme activity competitively, when added to egg PC substrate. The inhibition of LCAT activity was not due to changes in the fluidity of the substrate particle. However, the inhibition depended on the position occupied by TUFA in the PC, as well as on the paired fatty acid. Thus, for human LCAT, 18:1t was more inhibitory when present at sn-2 position of PC, than at sn-1, when paired with 16:0. In contrast, when paired with 20:4, 18:1t was more inhibitory at sn-1 position of PC. Both human and rat LCATs, which are normally specific for the sn-2 acyl group of PC, exhibited an alteration in their positional specificity when 16:0-18:1t PC or 16:1t-20:4 PC was used as substrate, deriving 26-86% of the total acyl groups for cholesterol esterification from the sn-1 position. These results show that the trans fatty acids decrease high density lipoprotein through their inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, and also alter LCAT's positional specificity, inducing the formation of more saturated cholesteryl esters, which are more atherogenic.

Impaired function of lecithin:cholesterol acyltransferase in atherosclerosis-susceptible White Carneau pigeons: possible effects on metabolism of oxidized phospholipids.

Although White Carneau (WC) pigeons are known to be more susceptible to atherosclerosis than Show Racer (SR) pigeons, the reasons for this difference are not fully understood. While no major differences are known in the lipoprotein composition, a difference in the cholesteryl ester (CE) composition was reported. However, there is little information on the activity or specificity of lecithin:cholesterol acyltransferase (LCAT), the major source of plasma CE. In order to determine whether the esterification of cholesterol or other functions of LCAT are compromised in WC pigeons, we studied the various reactions catalyzed by LCAT in the two groups. The cholesterol esterification was found to be significantly lower in WC pigeons, whether assayed with exogenous or endogenous substrates. Furthermore, lyso phosphatidylcholine (PC) esterification and oxidized PC hydrolysis, two other reactions carried out by LCAT, were also lower in WC. We found evidence for the presence of an active lysophospholipase in pigeon plasma, and this activity was also lower in WC compared to SR. A significant increase in the FC/PC ratio, another reported atherogenic risk factor, was found in WC. plasma. Because of the absence of other hydrolytic enzymes in pigeon plasma, LCAT may play an important role in the metabolism of oxidized PC generated during lipoprotein oxidation, and therefore a decrease in its activity in White Carneau pigeons may contribute to increased risk of atherosclerosis.

Novel function of lecithin-cholesterol acyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoprotein oxidation.

Although the major function of lecithin-cholesterol acyltransferase (LCAT) is cholesterol esterification, our previous studies showed that it can also hydrolyze platelet-activating factor (PAF). Because of the structural similarities between PAF and the truncated phosphatidylcholines (polar PCs) generated during lipoprotein oxidation, we investigated the possibility that LCAT may also hydrolyze polar PCs to lyso-PC during the oxidation of plasma. PAF acetylhydrolase (PAF-AH), which is known to hydrolyze polar PCs in human plasma, was completely inhibited by 0.2 mM p-aminoethyl benzenesulfonyl fluoride (Pefabloc), a new serine esterase inhibitor, which had no effect on LCAT at this concentration. On the other hand, 1 mM diisopropylfluorophosphate (DFP) completely inhibited LCAT but had no effect on PAF-AH. Polar PC accumulation during the oxidation of plasma increased by 44% in the presence of 0.2 mM Pefabloc and by 30% in the presence of 1 mM DFP. The formation of lyso-PC was concomitantly inhibited by both of the inhibitors. The combination of the two inhibitors resulted in the maximum accumulation of polar PCs, suggesting that both PAF-AH and LCAT are involved in their breakdown. Oxidation of chicken plasma, which has no PAF-AH activity, also resulted in the formation of lyso-PC from the hydrolysis of polar PC, which was inhibited by DFP. Polar PCs, either isolated from oxidized plasma or by oxidation of labeled synthetic PCs, were hydrolyzed by purified LCAT, which had no detectable PAF-AH activity. These results demonstrate a novel function for LCAT in the detoxification of polar PCs generated during lipoprotein oxidation, especially when the PAF-AH is absent or inactivated.

Impaired cholesterol esterification in the plasma in patients with breast cancer.

An important factor which determines the movement of cholesterol in and out of the cells is the free cholesterol (FC)/esterified cholesterol (EC) ratio in the plasma. Although this ratio has been shown to be increased in several types of malignancies in humans as well as experimental animals, it is not known whether such an abnormality is found in breast cancer patients. Furthermore, the reasons for such an increase in cancer patients are unknown. We studied the plasma lipid composition and the activity of lecithin-cholesterol acyltransferase (LCAT), the enzyme responsible for the formation of most of EC in human plasma, in 12 women with breast cancer and 9 age-matched control women. The plasma EC concentration was found to be significantly decreased in cancer patients, whereas the FC concentration was unchanged, leading to increased FC/EC ratios (P < 0.05). The concentration of phosphatidylcholine, the acyl donor in the LCAT reaction, was decreased significantly, whereas all other phospholipids were unaffected. The cholesterol-esterifying activity of LCAT was significantly lower in cancer patients, whether assayed with endogenous substrates (P < 0.05), or with an exogenous substrate (P < 0.01). However, another function of the enzyme, namely the lysolecithin acyltransferase activity, was increased (P < 0.02), indicating that the enzyme concentration in plasma may not be decreased. These results show that the increase in the FC/EC ratio in cancer patients is due to an impaired esterification of cholesterol by plasma LCAT, probably due to an alteration in the composition of substrate lipoproteins, or the presence of an inhibitory factor.

Analysis of human lecithin-cholesterol acyltransferase activity by carboxyl-terminal truncation.

Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (approximately 50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate, p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.

Disparate effects of oxidation on plasma acyltransferase activities: inhibition of cholesterol esterification but stimulation of transesterification of oxidized phospholipids.

Oxidation of lipoproteins results in the formation of several polar phospholipids with pro-inflammatory and pro-atherogenic properties. To examine the possible role of lecithin/cholesterol acyltransferase (LCAT) in the metabolism of these oxidized phospholipids, we oxidized whole plasma with either Cu(2+) or a free-radical generator, and determined the various activities of LCAT. Oxidation caused a reduction in plasma phosphatidylcholine (PC), an increase in a short-chain polar PC (SCP-PC), and an inhibition of the transfer of long-chain acyl groups to cholesterol (LCAT activity) or to lyso PC (lysolecithin acyltransferase (LAT) I activity). However, the transfer of short-chain acyl groups from SCP-PC to lyso PCLAT II activity) was stimulated several fold, in direct correlation with the degree of oxidation. LAT II activity was not stimulated by oxidation in LCAT-deficient plasma, showing that it is carried out by LCAT. Oxidized normal plasma exhibited low LCAT activity even in the presence of exogenous proteoliposome substrate, indicating that the depletion of substrate PC was not responsible for the loss of activity. Oxidation of isolated LDL or HDL abolished their ability to support LCAT and LAT I activities of exogenous enzyme, but promoted the LAT II activity. Purified LCAT lost its LCAT and LAT I functions, but not its LAT II function, when oxidized in vitro. These results show that while oxidation of plasma causes a loss of LCAT's ability to transfer long-chain acyl groups, its ability to transfer short-chain acyl groups, from SCP-PC is retained, and even stimulated, suggesting that LCAT may have a physiological role in the metabolism of oxidized PC in plasma.

Marine lipids normalize cholesteryl ester transfer in IDDM.

Patients with insulin-dependent diabetes mellitus (IDDM) have a pathological increase in cholesteryl ester transfer (CET) that enriches the apolipoprotein B-containing lipoproteins with cholesteryl ester and increases their atherogenicity. Since we have shown earlier that omega-3 (n-3) fatty acids present in marine lipids normalize both CET and lipoprotein composition in non-diabetic patients with hypercholesterolaemia, we sought to determine whether the same beneficial effects could be achieved in nine normolipidaemic (triglycerides 1.10; cholesterol 4.94, high density lipoprotein 1.10 mmol/l) IDDM patients (fructosamine 424 +/- 156; normal 174-286 mumol/l) treated for 2 months with n-3 fatty acids (4.6 g/day). Before treatment, CET measured by both mass and isotopic assays was abnormally accelerated (p < 0.001). While marine lipids modestly decreased triglyceride levels (-14%; p < 0.05 ), CET fell dramatically in all subjects (mass assay: -97% at 1 h; isotopic assay: -58%; p < 0.001) to below control levels with no change in glycaemic control (fructosamine 408 +/- 103 mumol/l). The mass of cholesteryl ester transfer protein paradoxically increased significantly (pre-treatment: 2.04 +/- 0.86 vs post-treatment 2.48 +/- 0.97 micrograms/ml; p < 0.05). Since it is believed that accelerated CET promotes the formation of atherogenic cholesteryl ester-enriched apo B-containing lipoproteins, the capacity of marine lipids to reverse this functional abnormality without altering glycaemic control suggests that these agents may have an adjunctive role to play in the nutritional therapy of IDDM.

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates.

Comparative studies indicate that plasma cholesteryl ester (CE) composition is correlated with susceptibility to atherosclerosis. We previously showed that the lecithin:cholesterol acyltransferases (LCATs) of susceptible species such as rabbit, pig, and chicken (group I) differ in their substrate and positional specificities from the LCATs of resistant species such as rat and mouse (group II). However, the relative importance of enzyme specificity and substrate phosphatidylcholine (PC) composition in determining the CE composition is not known. To address this, we analyzed the molecular species composition of plasma PC in the same 14 vertebrates in which we previously studied the CE composition and LCAT specificity. The utilization of native PC species by LCAT was studied by determining the loss of each PC after incubation of plasma at 37 degrees C. The major contributor for LCAT reaction was either 16:0-18:2 PC or 18:0-18:2 PC in all species except dog, in which it was 18:0-20:4 PC. The formation of 20:4 CE correlated more with the consumption of 18:0-20:4 PC in group I, and with the consumption of 16:0-20:4 PC in group II. The group II enzymes exhibited higher selectivity for sn-2-20:4 PCs, whereas the group I enzymes showed preference for sn-2-18:2 PCs. The synthesis of high percentage of 20:4 CE in dog plasma was found to be due to the presence of unusually high concentration of 18:0-20:4 PC, rather than due to enzyme selectivity. These results show that the PC molecular species composition, especially the concentrations of sn-2-20:4 phosphatidylcholines has profound influence on plasma CE composition, and possibly on atherogenic risk.

Specificity of lecithin:cholesterol acyltransferase and atherogenic risk: comparative studies on the plasma composition and in vitro synthesis of cholesteryl esters in 14 vertebrate species.

To determine whether the specificity of lecithin: cholesterol acyltransferase (LCAT) influences the susceptibility to atherosclerosis, we compared the composition and in vitro synthesis of cholesteryl ester (CE) in the plasmas of 14 vertebrate species with varying predisposition to atherosclerosis. The susceptible species (Group I) had significantly higher ratios of 16:0 CE/20:4 CE in their plasma than the resistant species (Group II). The in vitro formation of labeled CE species in native plasma from labeled cholesterol correlated highly with the mass composition, showing that the LCAT reaction is the predominant source of plasma CE in all the animal species examined. Isolated LCATs from Group I species also synthesized CE with higher ratios of 16:0/20:4 than LCATs from Group II when egg phosphatidylcholine (PC) was used as the acyl donor. In addition, the Group I LCATs exhibited lower specificity towards sn-2-20:4 and sn-2-22:6 PCs, and higher specificity towards sn-2-18:2 PC species than Group II LCATs. With 16:0-20:4 PC as the substrate, all Group I LCATs synthesized more 16:0 CE than 20:4 CE, whereas all Group II LCATs, with the exception of dog enzyme, synthesized predominantly 20:4 CE, showing that the two types of LCAT have different positional specificities towards this PC. These results suggest that there are two classes of LCAT in nature that differ from each other in their substrate and positional specificities, possibly because of differences in their active-site architectures. We propose that the presence of one type of LCAT, which cannot efficiently transfer certain long chain polyunsaturated acyl groups and which consequently synthesizes more saturated CE, may increase the risk of atherosclerosis.

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts.

To better understand the mode of action of omega 3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 microM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which > 95% was associated with phospholipids. In addition, the concentration of 22:5n-3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P < 0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P < 0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P < 0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5'-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P < 0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n-3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.

Role of sn-2 acyl group of phosphatidylcholine in determining the positional specificity of lecithin-cholesterol acyltransferase.

Although human plasma lecithin-cholesterol acyltransferase (LCAT) is believed to be specific for the sn-2 position of phosphatidylcholine (PC), our recent studies showed that it derives a significant percent of acyl groups from the sn-1 position of certain PC species. To understand the physicochemical basis for this altered positional specificity, we determined the effect of sn-2 acyl group of PC on the enzyme activity and utilization of 16:0 from the sn-1 position by purified human and rat LCATs. Positional isomers of PC containing 16:0 at sn-2 were better substrates for human LCAT than the corresponding sn-1-16:0 isomers, whereas the reverse was true for rat LCAT. The positional specificity of human LCAT varied greatly depending on the nature of the acyl group at sn-2. The sn-1 contribution from various sn-1-16:0-2-acyl PCs for cholesteryl ester (CE) synthesis was 1.0% from 16:0-16:0, 1.4% from 16:0-20:5, 7.3% from 16:0-18:1, 47.0% from 16:0-20:3, 49.9% from 16:0-20:4, 54.9% from 16:0-22:6, and 72.3% from 16:0-18:0. There was a linear relationship between the percentage of 16:0 CE formed (from sn-1 position) and the acyl chain length at sn-2 position (r = 0.94). Rat LCAT also transferred some 16:0 from sn-1 position of 16:0-22:6, 16:0-20:3, and 16:0-18:0 PCs, but not from the other natural PCs tested. The phospholipase A activity of both LCATs in the presence of 16:0-20:4 PC showed the same positional specificity as CE synthesis, indicating that the specificity is determined at the formation of acyl-enzyme intermediate. These results show that the positional specificity of LCAT is influenced by the structure of PC, especially the chain length of the sn-2 acyl group.