Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E° mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 – 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7β-hydroxycholesterol (7β-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E° mice, whereas the level of 7α-hydroxycholesterol (7α-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E° mice.     

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E degrees mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 - 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7beta-hydroxycholesterol (7beta-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E degrees mice, whereas the level of 7alpha-hydroxycholesterol (7alpha-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E degrees mice.     

Particle-size distribution of very low density plasma lipoproteins during fat absorption in man

The size distributions of electrophoretically isolated subfractions of the very low density human plasma lipoproteins have been determined using electron microscopy. The primary and secondary particles observed in plasma of normal subjects after fat ingestion appear to have similar size distributions. Particles produced by corn oil feeding can be fixed by the osmium tetroxide reaction while those produced by butter fat feeding could not be fixed or made visible by this technique. Good agreement between particle size as measured by electron microscopy and particle size as predicted by ultra-centrifugal analysis was obtained.     

Macrocyclic lactones: distribution in plasma lipoproteins of several animal species including humans

We studied the in vitro distribution of macrocyclic lactones (MLs), lipophilic anthelmintic drugs, in the plasma of several animal species including humans. First, in vitro spiking of goat plasma was performed with ivermectin, moxidectin, abamectin, doramectin, or eprinomectin. In parallel, goats were treated with subcutaneous injection of ivermectin. Then, cow, sheep, rabbit, pig, and human plasma were spiked with moxidectin. Four fractions were separated using KBr density gradient ultracentrifugation: very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein-deficient fraction. Cholesterol was analyzed by enzymatic assay and MLs by high-performance liquid chromatography. An average of 96% of MLs was associated with lipoproteins. The five MLs studied distributed similarly into goat plasma fractions with a preferential association with HDL (80-90%). Ivermectin partitioning in goat plasma was similar after in vitro spiking and in vivo treatment. In species displaying various lipoprotein profiles, moxidectin was also mainly associated with HDL. However, in human plasma, moxidectin was associated with a lesser extent to HDL (70%) and more to LDL (22%) when compared to other animal species. A relation between the plasma cholesterol content and pharmacokinetics of the drug is suggested. Our finding will allow further exploration of intestinal lymphatic absorption and milk elimination of these compounds-mechanisms in which lipoproteins are involved. In addition, possible improvements of new drug delivery systems are suggested.     

Rapid on-line determination of cholesterol distribution among plasma lipoproteins after high-performance gel filtration chromatography

A high-performance gel chromatography (HPGC) system has been developed which allows the unattended on-line determination of lipoprotein cholesterol distribution (VLDL-C, LDL-C, HDL-C), within 40 min, in microliter quantities of plasma using a single, relatively inexpensive column (Superose 6HR). The FAST cholesterol reagent (Sclavo) and a knitted PFTE Kratos reaction coil (Applied Biosystems) were found to provide optimal sensitivity, linearity, resolution, and dispersion characteristics. Validation is provided by comparison to target values for human quality control reference sera, and by comparing the values obtained by HPGC to the beta-quant method (LRC). The utility of the system is illustrated by comparing profiles from seven different species with normal or elevated plasma cholesterol concentrations. This technique allows rapid analysis of samples, regardless of species, without the use of precipitating agents or the ultracentrifuge. It could also be applied for the direct clinical determination of LDL-cholesterol.     

Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus

Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 associated with lipoproteins that hydrolyzes platelet-activating factor (PAF) and oxidized phospholipids. We have developed an ELISA for PAF-AH that is more sensitive than previous methods, and have quantified HDL-associated and non-HDL-associated PAF-AH in healthy, hyperlipidemic, and diabetic subjects. In healthy subjects, plasma total PAF-AH concentration was positively correlated with PAF-AH activity and with plasma total cholesterol, triacylglycerol, LDL cholesterol and apolipoprotein B (apoB) concentrations (all P < 0.01). HDL-associated PAF-AH concentration was correlated positively with plasma apoA-I and HDL cholesterol. Subjects with hyperlipidemia (n = 73) and diabetes mellitus (n = 87) had higher HDL-associated PAF-AH concentrations than did controls (P < 0.01). Non-HDL-associated PAF-AH concentration was lower in diabetic subjects than in controls (P < 0.01). Both hyperlipidemic and diabetic subjects had lower ratios of PAF-AH to apoB (P < 0.01) and higher ratios of PAF-AH to apoA-I (P < 0.01) than did controls. Our results show that the distribution of PAF-AH mass between HDLs and LDLs is determined partly by the concentrations of the lipoproteins and partly by the mass of enzyme per lipoprotein particle, which is disturbed in hyperlipidemia and diabetes mellitus.     

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1^−/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1^−/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1^−/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1^−/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1^−/−, apoE^−/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1^−/−, apoE^−/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.     

Disc electrophoresis of rat plasma lipoproteins

The disc electrophoresis of lipoproteins of unfractionated rat plasma is described. The plasma was prestained with Sudan black B and electrophoretically separated at polyacrylamide gel concentrations of 7.5, 5, and 3.75%. At least four lipoprotein components were observed, and an additional 2-3 components in the main gel and 2-5 components in the spacer gel possibly were present. Densitometry of the resolved gel patterns indicated good reproducibility. Thin-layer chromatography of lipids extracted from the Sudan black B-binding components confirmed the lipoprotein nature of these components of rat plasma. A comparison of the disc electrophoretic patterns of human serum and rat plasma suggested that the low-density lipoprotein components of rat plasma are smaller in size than those of human serum.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Apolipoprotein E distribution among human plasma lipoproteins: role of the cysteine-arginine interchange at residue 112

Human apolipoprotein (apo) E occurs as three common isoforms (apoE4, E3, and E2), all of which influence plasma cholesterol levels. Although both apoE4 and E3 bind with equal effectiveness to the low density lipoprotein receptor, they associate preferentially with different classes of plasma lipoproteins: apoE4 with very low density lipoproteins, apoE3 with high density lipoproteins. The primary structure of apoE3 differs from that of apoE4 at only a single site; apoE3 has its sole cysteine residue at position 112, while apoE4 contains arginine at position 112 and completely lacks cysteine. The present study investigated how this structural difference between apoE4 and E3 determines their distribution among plasma lipoproteins, and analyzed the role of the disulfide-linked heterodimer apoE-A-II (which apoE4 cannot form) in determining the distribution. Human plasma was incubated with 125I-labeled apoE, and lipoproteins were separated by agarose chromatography. Both apoE3 that had been reduced and alkylated with iodoacetamide and apoE3-A-II distributed with high density lipoproteins, indicating that a combination of an inherent property of the monomeric apoE3 structure and apoE-A-II formation account for distribution of apoE3 to the high density lipoproteins. Cysteamine modification of apoE3 resulted in an apoE4-like distribution, demonstrating that a positive charge at position 112 determined the apoE4 distribution and that the effect was not exclusively due to the presence of arginine at this position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol turnover in swine plasma lipoproteins

The kinetics of free and esterified cholesterol labeling were studied in the plasma lipoproteins of three groups of six adult Large White sows, after either an intravenous injection of autologous red cells previously labeled with [3H]-cholesterol, an intravenous injection of [14C]-acetate, or a [14C]-cholesterol labeled meal. The specific radioactivities became equal in plasma and red cell cholesterol about 96 hours after each pulse of radioactive cholesterol. This finding indicates that red cell cholesterol is completely exchangeable in vivo, with a turnover time of 8.5 hours. The VLDL were shown to play a preferential role in the transport in the plasma of newly synthetized cholesterol. This role is shared with chylomicrons in the transport of absorbed dietary cholesterol, which appears in the plasma mainly as esterified cholesterol. Cholesteryl esters of VLDL are not the main source for those of LDL, which could be labeled by intraplasmatic exchanges or transfers of esterified cholesterol.     

The interaction of human plasma glycoaminoglycans with plasma lipoproteins.

Modifications of existing methods have allowed for the isolation and purification of various species of plasma glycosaminoglycans on the basis of their sulfate content and molecular size. All of the preparations precipitated human plasma low density lipoproteins (LDL); maximal precipitation occurred with amounts of glycans corresponding to 50 mug of hexuronate and 12 mg of LDL. The interaction of glycans with pyrene-labeled lipoproteins was also studied, measuring variations of the fluorescence emitted by the monomer (M) and excimer (E) species of the bound pyrene. The ratio IE/IM is proportional to c/eta, where c is the microscopic concentration of the pyrene confined to the hydrocarbon region of the lipoprotein and eta is the microviscosity of that region. To 0.12 mg of pyrene-labeled LDL, very low density lipoproteins (VLDL) or high density lipoproteins (HDL) were added increasing amounts of the various glycan preparations. The sulfate-rich species decreased the IE/IM ratio of LDL and HDL but not that of VLDL. This finding suggests that the glycan caused a change in lipoprotein conformation associated with either an increased volume or increased microscopic viscosity of the hydrocarbon region. The modification of LDL conformation could be prevented by proteolytic treatment of the sulfate-rich species or by addition to the system of suitable amounts of sulfate-poor species or of chrondroitin-4-sulfate, but could not be prevented by increased ionic concentration. These results suggest that the two main species of plasma glycans are important in maintaining adequate rheological properties of plasma lipoproteins.     

Association of antisense oligonucleotides with lipoproteins prolongs the plasma half-life and modifies the tissue distribution.

In order to direct antisense oligonucleotides to specific tissues or cell types in vivo, we are exploring the possibility to utilize lipoproteins as transport vehicles. A 16-mer oligonucleotide (ODN) was derivatized at the 5' prime through a 32P phosphate spacer with cholesterol, yielding a 32P-labeled amphiphatic cholesteryl-oligonucleotide (cholODN). Incubation of cholODN with low-density lipoprotein (LDL) for 2 hr at 37 degrees C resulted in the formation of a cholODN-LDL complex that migrates as a single peak on agarose gel electrophoresis. The cholODN was found to bind quantitatively to both high-density lipoproteins (HDL) and LDL, but not to albumin. Stable oligonucleotide-LDL particles with up to 50 molecules of cholODN per LDL particle could be obtained. In contrast, the control ODN did not show affinity for plasma lipoproteins. Upon injection into rats, cholODN became rapidly associated with plasma lipoproteins while control ODNs were recovered in the lipoprotein deficient serum fraction. The plasma half-life of cholODN (9-11 min) is considerably prolonged as compared with the control ODN (t1/2 less than 1 min). The cholODN-LDL was at least 5 min stable against degradation by rat plasma nucleases. It is concluded that derivatization of antisense oligonucleotides with cholesterol profoundly modifies their in vivo fate and opens possibilities for efficient and specific receptor-dependent targeting, mediated by lipoproteins coupled with specific recognition markers to various hepatic cell types.