Cholesterol flux between lipid vesicles and apolipoprotein AI discs of variable size and composition

Reconstituted discoidal high-density lipoproteins (rHDLs) of apolipoprotein AI are able to induce leakage of the internal aqueous space of lipid vesicles (A. Tricerri et al., 1998, Biochim. Biophys. Acta 1391, 67-78) and such interaction depends on the cholesterol content of vesicles and rHDL as well as the rHDL size. With the aim of knowing if this rHDL/vesicle interaction plays some role in the cholesterol exchange, the time course for bidirectional radiolabeled cholesterol transfer between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles and different sized rHDLs was measured. The results show that size increase in the rHDL decreases the rate constant for cholesterol transfer from POPC/cholesterol vesicles and that the initial presence of cholesterol in the vesicles results in an increased rate constant for cholesterol transfer from the rHDLs. This cannot be explained by a simple aqueous diffusion mechanism. The existing correlation between rHDL/vesicle interaction and cholesterol transfer rate suggests that besides the aqueous diffusion, another mechanism involving the binding or interaction between donor and acceptor may occur. This fact may be of physiological relevance since the relative high affinity of small cholesterol-poor discs for cell membranes could facilitate the cholesterol efflux, while the decreased membrane affinity as a consequence of cholesterol enrichment and increase in size would decrease the rate of transfer in the opposite direction.     

Interaction of alpha-tocopherol with model human high-density lipoproteins.

The effects of alpha-tocopherol on the properties of model high-density lipoproteins (HDLs), composed of human apolipoprotein A-I and dimyristoylphosphatidylcholine, were investigated by physicochemical methods. The intrinsic fluorescence of alpha-tocopherol and its effects on the polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, which probes the hydrocarbon region of the lipids, and 4-heptadecyl-7-hydroxycoumarin, which is a probe of lipid surfaces, suggest that alpha-tocopherol is located at the lipid-water interface. Relative to cholesterol, alpha-tocopherol in lipid surfaces is virtually inert physicochemically. Incorporation of alpha-tocopherol into HDLs induces only a modest increase in particle size, no change in the transition temperature, and little change in lipid polarity and lipid-lipid interactions. Moreover, alpha-tocopherol has only a negligible effect on the kinetic parameters of the lipophilic enzyme lecithin:cholesterol acyltransferase, which binds to phosphatidylcholine surfaces and forms cholesteryl esters. However, alpha-tocopherol has a dramatic inhibitory effect on the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine, a process that occurs through the insertion of the protein into preformed defects in the lipid surface. It is proposed that alpha-tocopherol inhibits the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine by inserting into defects within the lipid surface, thereby reducing the size and/or number of sites for insertion of apolipoprotein A-I.     

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.     

Apolipoprotein C-I binds more strongly to phospholipid/triolein/water than triolein/water interfaces: a possible model for inhibiting cholesterol ester transfer protein activity and triacylglycerol-rich lipoprotein uptake

Apolipoprotein C-I (apoC-I) is an important constituent of high-density lipoprotein (HDL) and is involved in the accumulation of cholesterol ester in nascent HDL via inhibition of cholesterol ester transfer protein and potential activation of lecithin:cholesterol acyltransferase (LCAT). As the smallest exchangeable apolipoprotein (57 residues), apoC-I transfers between lipoproteins via a lipid-binding motif of two amphipathic α-helices (AαHs), spanning residues 7-29 and 38-52. To understand apoC-I's behavior at hydrophobic lipoprotein surfaces, oil drop tensiometry was used to compare the binding to triolein/water (TO/W) and palmitoyloleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. When apoC-I binds to either interface, the surface tension (γ) decreases by ~16-18 mN/m. ApoC-I can be exchanged at both interfaces, desorbing upon compression and readsorbing on expansion. The maximal surface pressures at which apoC-I begins to desorb (Π(max)) were 16.8 and 20.7 mN/m at TO/W and POPC/TO/W interfaces, respectively. This suggests that apoC-I interacts with POPC to increase its affinity for the interface. ApoC-I is more elastic on POPC/TO/W than TO/W interfaces, marked by higher values of the elasticity modulus (ε) on oscillations. At POPC/TO/W interfaces containing an increasing POPC:TO ratio, the pressure at which apoC-I begins to be ejected increases as the phospholipid surface concentration increases. The observed increase in apoC-I interface affinity due to higher degrees of apoC-I-POPC interactions may explain how apoC-I can displace larger apolipoproteins, such as apoE, from lipoproteins. These interactions allow apoC-I to remain bound to the interface at higher Π values, offering insight into apoC-I's rearrangement on triacylglycerol-rich lipoproteins as they undergo Π changes during lipoprotein maturation by plasma factors such as lipoprotein lipase.     

The transport of alpha-tocopherol and beta-carotene in human blood.

The concentrations and distributions of major lipids (cholesterol, phospholipid, and triglyceride), tocopherol and carotenoids were determined in the plasma lipoprotein fractions (VLDL, LDL, and HDL) of (1) normal human subjects, (2) patients with hyperlipoproteinemia, and (3) patients with erythropoietic protoporphyria treated with oral beta-carotene and/or alpha-tocopherol. The distribution of tocopherol (in percent) was most closely correlated with the distribution of total lipids in the individual lipoproteins, while the major portion of beta-carotene was present in the low density lipoproteins, irrespective of the lipid distribution in the lipoproteins (except for one subject with hyperchylomicronemia). The alpha-tocopherol and beta-carotene concentrations of plasma and RBC in patients treated with tocopherol and carotene were determined periodically for a one-year period. Plasma and RBC tocopherol concentrations showed a rapid, parallel increase in response to tocopherol supplementation. In contrast, the plasma and RBC carotene concentrations showed a much slower and nonparallel increase in response to carotene administration. When carotene supplementation was stopped, the elevated carotene levels in both plasma and RBC persisted for several months; the elevated plasma carotene level persisted longer than the raised RBC carotene levels. These results suggest that alpha-tocopherol and beta-carotene are transported differently in the circulation and that the tissue storage and mobilization of these compounds are different.     

Relationships between serum levels of autoantibodies against oxidized low density lipoproteins, lipid-soluble antioxidants and apolipoprotein B in patients with coronary heart disease

High affinity IgG autoantibodies against oxidized low density lipoproteins (oxLDLs), apolipoprotein B and lipid-soluble antioxidants--alpha-tocopherol and beta-carotene, were tested in patients with coronary heart disease. Correlation relationships between these parameters were analysed. Fifty one patients with coronary heart disease (37 males/14 females) defined as Q-wave myocardial infarction and/or stenosis of more than 50%, and 51 healthy blood donors (34 males/17 females) as controls participated in this study. LDLs were isolated by density gradient ultracentrifugation and oxidized with Cu2+. OxLDLs or native LDLs (nLDLs) were used as antigens in enzyme immunoassay (ELISA) to detect IgG autoantibodies in the serum. The contents of alpha-tocopherol and beta-carotene were measured by HPLC. Apolipoprotein B was determined by immunoturbidimetry. Correlation analysis of the parameters was carried out by Spearmann's test. Alpha-tocopherol was decreased significantly in the serum of patients with coronary heart disease (2.96+/-1.63 nmol/mg serum protein vs 6.23+/-2.28 nmol/mg serum protein in Control group) (p < 0.01). Also, the serum level of beta-carotene was decreased in patients with coronary heart disease (174.0+/-95.7 pmol/mg serum protein vs 313.2+/-141.5 pmol/mg serum protein in Control group) (p < 0.01), while apolipoprotein B was increased significantly (1.20+/-0.34 g/l in patients with coronary heart disease vs 0.86+/-0.23 g/l in Control group) (p < 0.001). In a previous study we established that the mean serum level of IgG autoantibodies against oxLDLs (expressed in optical density units) was about 2.5 times higher in patients with coronary heart disease as compared to control subjects (p < 0.001). A good positive linear correlation was observed between alpha-tocopherol and apolipoprotein B levels in Control group (r = 0.78, p < 0.001), as well as in the group of patients with coronary heart disease (r = 0.42, p < 0.001). Poor nonsignificant correlations were established between all another measured parameters. In conclusion, the lipid-soluble antioxidants--alpha-tocopherol and beta-carotene, are not informative with respect to the susceptibility of the serum to oxidative modifications and as to the extent of the subsequent humoral immune response. Presumably, the reduction of the correlation coefficient between apolipoprotein-B and alpha-tocopherol in patients with coronary heart disease in comparison with control subjects could provide indirect information on modifications of apolipoprotein-B and on a decrease of its susceptibility to interact with this major lipid-soluble antioxidant in atherogenesis.     

Effects of hydrophobicity on turnover of plasma high density lipoproteins labeled with phosphatidylcholine ethers in the rat

Rat high density lipoproteins (HDL) were labeled with a series of phosphatidylcholines and ether analogs of phosphatidylcholine. The rates of turnover of the phosphatidylcholine ethers in the rat decreased as a function of increasing hydrophobicity and were more than five times faster than those of apolipoprotein A-I turnover and spontaneous lipid transfer. The major tissue sites for uptake were the liver, adrenals, and ovaries. The rate of turnover of a phosphatidylcholine was faster than that of the corresponding ether analog due to the action of lecithin:cholesterol acyltransferase, although this activity was slow compared to the turnover of high density lipoprotein-phosphatidylcholine. Injection of a purified human phosphatidylcholine transfer protein increased the turnover rate of a phosphatidylcholine and its ether analog. We conclude that a major route for the turnover of plasma high density lipoprotein-phosphatidylcholine in the rat is independent of spontaneous lipid transfer, hydrolysis, and HDL particle uptake, and that it involves the activity of a plasma phosphatidylcholine transfer protein.     

Protection of endogenous beta-carotene in LDL oxidized by oxygen free radicals in the presence of supraphysiological concentrations of melatonin

This study was designed to evaluate the effect of high concentrations of melatonin on the peroxidation of human low density lipoproteins (LDLs) initiated by O(2)(*-) and ethanol-derived peroxyl radicals (RO(2)(*)) from water gamma radiolysis in the presence of ethanol. LDL (3 g/l; total LDL concentration) was oxidized in the absence of melatonin or in its presence at three concentrations (50 x 10(-6), 100 x 10(-6) or 250 x 10(-6) mol/l) in ethanol. Radiolytic yields (i.e. number of mole consumed or produced per Joule) of the markers of lipid peroxidation were determined (i.e. decrease in the endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances [TBARS]). Melatonin decreased the yields of lipid peroxidation products and delayed the onset of the propagation phase for conjugated dienes and TBARS in a concentration-dependent manner. Nevertheless, melatonin did not protect endogenous alpha-tocopherol against peroxyl-induced oxidation (probably due to a lower scavenging capacity than that of alpha-tocopherol towards peroxyl radicals), but delayed the consumption of LDL endogenous beta-carotene and decreased its rate of disappearance. The effect of melatonin seemed to be the highest for a melatonin concentration of 250 x 10(-6) mol/l.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.     

Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.     

Effects of blocking plasma lipid transfer protein activity in the rabbit

Plasma lipid transfer protein activity was completely blocked in rabbits for up to 48 h by infusion with goat antibody to rabbit lipid transfer protein. Lipid transfer protein activity in plasma of control animals, infused with antibody from a non-immune goat, decreased during the experiment but was never less than 50% of pre-infusion levels. During the period that lipid transfer protein activity was completely blocked, there were changes in high-density lipoprotein composition (expressed as % by weight) with a reduction in triacylglycerol from 8.4 +/- 2.4% to 1.0 +/- 0.2% (P less than 0.05) and an increase in esterified cholesterol from 10.7 +/- 1.7% to 14.5 +/- 0.3% (P less than 0.1). In conjunction with the observed changes in high-density lipoprotein composition, there was an increase in high-density lipoprotein particle size from a mean radius of 4.7 to 5.4 nm. The change in composition and particle size was not observed in high-density lipoproteins from control animals. There was a change in the distribution of plasma cholesterol in control animals, with a fall in the proportion of cholesterol in high-density lipoproteins (P less than 0.02) and consequently an increase in the proportion of cholesterol in low-density lipoproteins (P less than 0.02). However, the distribution of plasma cholesterol in animals in which lipid transfer protein activity was inhibited was maintained at original levels during the period of inhibition. Consequently, in these animals, there was a less atherogenic distribution of cholesterol during the period of lipid transfer protein inhibition when compared with control animals. The changes observed in lipoproteins, in the absence of lipid transfer protein activity, demonstrate that lipid transfer protein modifies lipoproteins in vivo and appears to contribute to a more atherogenic lipid profile.     

The inhibition of radical-initiated peroxidation of microsomal lipids by both alpha-tocopherol and beta-carotene.

Rat liver microsomal lipids in hexane solution were exposed to the lipid-soluble radical initiator, azobis-isobutyronitrile (AIBN), and the antioxidant activities of alpha-tocopherol and beta-carotene have been compared. Lipid peroxidation was monitored both by conjugated diene formation at 233 nm, and by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm. Diene formation was continuous for at least 120 min in the presence of 85 micrograms/ml lipid and 4 mM AIBN. Both alpha-tocopherol and beta-carotene acted as chain-breaking antioxidants, suppressing lipid peroxidation and producing an induction period at concentrations as low as 0.5 and 8 microM, respectively. When both of these lipid-soluble antioxidants were present together, the oxidation was strongly suppressed and the induction period was the sum of the individual antioxidants, alpha-Tocopherol and beta-carotene also inhibited MDA generation. In the presence of 170 micrograms/ml lipid and 8 mM AIBN, beta-carotene exhibited an IC50 of 1.1 microM and inhibited completely at 15 microM. Using beta-carotene, an induction period was observed, although much less pronounced than with alpha-tocopherol. Furthermore, beta-carotene inhibited MDA production in a concentration-dependent manner and exhibited an IC50 of 50 microM. In addition, added beta-carotene delayed the radical-initiated destruction of the endogenous alpha-tocopherol and gamma-tocopherol in this system.     

Interplay of oxygen, vitamin E, and carotenoids in radical reactions following oxidation of Trp and Tyr residues in native HDL3 apolipoproteins. Comparison with LDL. A time-resolved spectroscopic analysis

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.     

Influence of dietary vitamin E on the intermembrane transfer of alpha-tocopherol as mediated by an alpha-tocopherol binding protein

The effect of dietary vitamin E on the intermembrane transfer of (3R)-alpha-tocopherol, a spontaneous process accelerated in the presence of an alpha-tocopherol binding protein (alpha TBP), was examined. The transfer activity of this cytosolic liver protein was assayed via in vitro transfer of (3R)-alpha-[3H]tocopherol (alpha[3H]T) from egg lecithin liposomes to human erythrocyte ghosts (EG). Male Fisher 344 rats (1 and 20 months old) were fed diets containing 0, 30, and 500 mg/kg vitamin E (dl-alpha-tocopheryl acetate) for 15 weeks. Liver cytosol fractions were assayed for alpha[3H]T transfer activity (alpha TTA). Among young rats, those fed vitamin E-deficient diets had the highest alpha TTA, 5.02 +/- 3.10 pmole alpha[3H]T/min (mean +/- SD), which was different (P less than 0.05) from the spontaneous transfer rate of 2.10 pmole/min. Neither young rats fed 30 and 500 mg/kg vitamin E diets nor any of the aged rats showed alpha TTA which differed significantly from the spontaneous transfer rate. To examine the relationship between hepatic alpha-tocopherol levels and alpha TTA, alpha-tocopherol concentration per gram of wet liver was assayed by HPLC. A steep positive slope (6.39 +/- 1.46 pmole min-1 nmole g-1) and strong correlation (r = 0.873) between hepatic alpha-tocopherol and alpha TTA were observed (P less than 0.005) among young vitamin E-deficient rats. The data indicates that alpha TTA varies directly with hepatic alpha-tocopherol concentration when total liver vitamin E stores are very low. Thus, alpha TBP-mediated transfer of alpha-tocopherol may be manifest only when vitamin E status is compromised.     

Kinetics and mechanism of the spontaneous transfer of fluorescent phospholipids between apolipoprotein-phospholipid recombinants. Effect of the polar headgroup

Fluorescent derivatives of a phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and diacylglycerol have been studied to establish the effect of different polar headgroups on the mechanism and kinetics of spontaneous phospholipid transfer between recombinants of human plasma apolipoprotein A-II and dimyristoylphosphatidylcholine. The fluorescent lipids are all 1-myristoyl-2-[9-(1-pyrenyl)nonanoyl] glycerides. The transfer of the lipids is a first order process where the rate is independent of the concentration over a 50 fold range of the acceptor recombinants. These results are consistent with the lipids transferring as monomers being a water-soluble intermediate. The rate of transfer of the different phospholipids are slightly slower than phosphatidylcholine, with that of phosphatidylethanolamine being about 4 times slower. The transfer of phospholipids with a titratable headgroup is pH-dependent. The difference in the rates and pH dependence may be a function of the interactions (hydrogen bonding) between polar headgroups. The rate of transfer of the diacylglycerol is 20 times slower than phosphatidylcholine, but its activation energy (21 kcal/mol) is only 2 to 3 kcal less than most of the phospholipids (23 kcal/mol). These results suggest that the rate and activation energy for the spontaneous transfer of phospholipids can be predicted to a first approximation on the basis of its hydrophobic content, irrespective of the pH or identity of the polar headgroup.     

Influence of prostaglandins on the lipid transfer between human high density and low density lipoproteins

Prostaglandin (PG) E₁ was demonstrated to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins (HDL₃) to low density lipoproteins (LDL). The enhancement effect of PGE₁, on the interlipoprotein lipid transfer was seen at low PG concentrations under conditions of spontaneous exchange as well as in the presence of lipoprotein-depleted plasma, or partly purified plasma lipid exchange protein. PGE₂ and PGF₂ showed no significant influence on the interlipoprotein lipid transfer. Evidence is presented suggesting that the PGE₁-induced stimulation of interlipoprotein lipid exchange results in enhancement of LCAT-catalyzed cholesterol esterification in plasma. It is proposed that the effect of PGE₁ is due to the previously described PGE₁-induced reorganization of the HDL surface [(1984) FEBS Lett. 173, 291-293] and that PG-lipoprotein interaction may be a factor regulating cholesterol homeostasis.     

Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol

The distribution of apolipoprotein A-I, apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein in fasting normal human plasma was determined by two-dimensional electrophoresis followed by immunoblotting. The synthesis and transfer of labeled cholesteryl esters generated in plasma briefly incubated with [3H]cholesterol-labeled fibroblasts was followed in terms of the lipoprotein species containing these antigens. Following the early appearance of labeled free cholesterol in two pre beta-migrating apolipoprotein A-I species (Castro, G. R., and Fielding, C. J. (1988) Biochemistry 27, 25-29), labeled esters were first detected, after a 2-min delay, in a third pre beta-migrating species which also contained apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein. Pulse-chase experiments determined that label generated in this fraction was the precursor of at least a major part of labeled cholesteryl esters in the bulk of alpha-migrating high density lipoprotein. Over the maximum time course of these experiments (15 min, 37 degrees C), less than 10% of labeled cholesteryl esters were recovered in low or very low density lipoproteins separated by electrophoresis, immunoaffinity, or heparin-agarose chromatography. These data suggest channeling of cell-derived cholesterol and cholesteryl esters derived from it through a preferred pathway involving several minor pre beta-migrating lipoproteins to alpha-migrating high density lipoprotein.