A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein

The in vitro extraction of cholesterol from erythrocytes by plasma lipoproteins of reduced cholesterol content would be expected to be free of cholesterol-unrelated alterations of the cell membrane. The earlier application of this method utilized whole blood plasma in which the major part of the lipoprotein cholesterol was esterified by the plasma enzyme lecithin-cholesterol acyl transferase (LCAT) in a preliminary incubation. Because of the cholesterol remaining unesterified in the plasma, only 35% of the cell cholesterol could be removed. The method reported here uses HDL., a plasma lipoprotein which is the preferred substrate for LCAT, instead of whole plasma for the extraction. Multiple extractions with LCAT treated HDL, resulted in the removal of up to 77% of the erythrocyte cholesterol with only minor hemolysis.     

Effects of parasitism by the lung fluke,Haematoloechus medioplexus,on lung fatty acid and sterol composition in the bullfrog,Rana catesbiana

Gas-liquid chromatography was employed to determine cholesterol and total lipid fatty acid levels in lung tissue and blood from bullfrogs harboring natural infections of the lung fluke, Haematoloechus medioplexus. As compared with uninfected animals, infected hosts exhibited (1) negligible alterations in blood sterols and fatty acids, (2) consistently larger lungs, (3) a 40 per cent decrease in lung lipids, (4) a 50 per cent increase in lung cholesterol, and (5) little difference in mean values, but a higher variability in percentages of lung fatty acids. The parasite's fatty acid profile was more similar to that of host lung tissue than blood and exhibited a relatively high degree of unsaturation; cholesterol was the major sterol.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compensatory and adaptive changes in the small intestine and liver of rabbits after short-term cholesterol diet

Functional and morphological changes in the intestinal wall and liver were studied in rabbits on short-term cholesterol diet. It was established that with a rapid increase of cholesterol concentration in the general blood flow, the synthesis of high density lipoproteins in the intestinal wall was intensified. Enhanced hepatic elimination of cholesterol and chylomicrons from blood circulation contributes to cholesterol level stabilization in peripheral blood. With high density lipoprotein accumulation in the intestinal wall, cholesterol consumption did not change its concentration in the general blood flow. Structural changes in jejunal and liver mucosa were shown to depend on the degree of hypercholesterolemia and functional damage of these organs.     

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.     

Antihyperlipidaemic and antihypercholesterolaemic effects of Anethum graveolens leaves after the removal of furocoumarins.

Serum triacylglycerides and total cholesterol levels in rats, with hyperlipidaemia induced by diet, were determined after oral adminstration of a water extract of Anethum graveolens leaves before and after the extraction of the furocoumarin content of the leaves. Administration of the extracts consecutively for 14 days reduced the triacylglycerides and total cholesterol levels by almost 50 and 20%, respectively. Chloroform extraction of furocoumarins from the aqueous extracts did not reduce the antihyperlipidaemic potential of the extracts to a significant degree. Oral administration of the essential oil of A. graveolens seeds, at two different doses, also reduced the triacylglyceride levels by almost 42%. The total cholesterol level was not reduced by the same doses of the essential oil.     

The effect of blood serum on synthesis of lipids in rat thymocytes

The increase in the incorporation of [2-14C]sodium acetate into cholesterol, free fatty acids and rat thymocyte phospholipids during incubation of rat cells in the absence of blood serum was demonstrated. The label incorporation into the thymocytes was found to be activated in an insignificant degree. Rat blood sera obtained 1 hour after gamma-irradiation of animals with doses of 4 and 10 Gr contained the same concentrations of cholesterol and its esters as the intact rat sera. Irradiation of animals did not affect the ability of the sera to inhibit lipid synthesis in thymocytes or the levels of total phospholipids, cholesterol and its esters. Incubation of cells with the blood sera from irradiated rats led to an increase in the cholesterol/cholesterol ester ratio in the incubation mixture.     

Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin--a surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.     

Ontogeny of guinea pig liver cholesterol 7 alpha-hydroxylase: unexpected inverse correlation of enzyme activity with expanding bile acid pool

Changes in the activity of guinea pig liver cholesterol 7 alpha-hydroxylase (rate limiting enzyme of cholesterol catabolism) from birth to adult life was investigated using a microsomal acetone extraction method (to remove endogenous cholesterol). Contrary to the previously held notion, it was noted that while the total bile acid pool increased progressively with age after birth, hepatic cholesterol 7 alpha-hydroxylase specific activity declined. Neonatal hepatic cholesterol 7 alpha-hydroxylase showed an increase in enzyme activity in response to cell supernatant factors (100,000 xg supernatant) from neonatal livers, but not from adult livers.     

Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue.

In an experimental model of unilateral pneumonia caused by Klebsiella pneumoniae in rats we investigated whether intravenous administration of liposomes with prolonged blood circulation time resulted in significant localization of liposomes in infected lung tissue. Liposomes (100 nm) composed of hydrogenated phosphatidylinositol:hydrogenated phosphatidylcholine:cholesterol (molar ratio, 1:10:5) radiolabeled with gallium-67-deferoxamine showed relatively long blood circulation time. The degree of localization of these long circulating liposomes in the infected left lung was significantly higher compared to that of localization of 110 nm egg phosphatidylglycerol:egg phosphatidylcholine:cholesterol (molar ratio, 1:10:5) liposomes which exhibited relatively short blood circulation time. At 16 h after administration of the long circulating liposomes (when 10% of the injected dose was still present in the bloodstream) localization of liposomes in the infected left lung was increased up to 10-fold compared to the left lung of uninfected rats, and appeared to be highly correlated with the intensity of the infection. In the uninfected right lung the localization of long circulating liposomes was not increased. The degree of localization of liposomes in the infected tissue is dependent on the residence time of liposomes in the blood compartment. The extent of localization of long circulating liposomes in infected tissue appeared to be dependent on the liposomal dose administered.     

Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells

ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin—A surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-β-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-β-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.     

Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains.

BACKGROUND: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non-ionic detergents providing an easy analytical tool to study them. METHODS: Here, we applied a rapid detergent-based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. RESULTS: All investigated surface antigens of Niemann-Pick type C (NPC)-mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM-association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)-mutant monocytes with impaired cholesterol efflux did not show alterations in DRM-association. Differential CD14-dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM-association of the GPI-anchored proteins CD14, CD55, the Fcgamma receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM-association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcgamma RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. CONCLUSIONS: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis.     

Clustering of hemodynamic and metabolic abnormalities and of anthropometric characteristics in men and women, aged 21-60 years.

The associations between fat pattern and metabolic profile were evaluated, using cluster analysis, in 214 males and 244 females (aged 21-60 years). The subjects were randomly selected from the patients of one general practice in Castel D'Azzano (Italy). The metabolic status was indexed by blood pressure, blood glucose, uric acid, serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (both total and percentage). Fat pattern was evaluated using six skinfolds and three circumferences. The analyses were performed separately on anthropometric and metabolic variables for each sex. The analyses were repeated five times to evaluate the stability of the cluster solution. Anthropometric rather than metabolic variables provided a more stable solution for cluster analysis. Anthropometric clusters showed significantly different metabolic patterns. Most of the differences disappeared when a body mass index (BMI) effect was taken into account but not when an adjustment for waist to hip ratio (WHR) was made. On the other hand, neither BMI nor WHR was able to fully account for the differences in metabolic profiles of metabolic clusters. Cross-classification results of anthropometric and metabolic cluster membership showed a significant, although moderate, degree of association between the two classification sets. These results indicate that in both sexes anthropometric characteristics contribute to a limited degree to the metabolic profile and that the associations probably reflect a complex mechanism.     

Chemokine stimulation of lymphocyte alpha 4 integrin avidity but not of leukocyte function-associated antigen-1 avidity to endothelial ligands under shear flow requires cholesterol membrane rafts

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the alpha(4) integrins VLA-4 and alpha(4)beta(7) as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-beta-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of alpha(4)-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by G(i)-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although G(i)-dependent, was insensitive to raft disruption. Our results suggest that alpha(4) but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to G(i) signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct G(i) protein-associated machineries segregated within defined membrane compartments.     

High-temperature GC-MS-based serum cholesterol signatures may reveal sex differences in vasospastic angina

Alterations of cholesterol metabolism are responsible for vasospastic angina and atherosclerosis. To comprehensively evaluate cholesterol metabolism, 18 sterols, including cholesterol, 6 cholesteryl esters (CEs), 3 cholesterol precursors, and 8 hydroxycholesterols (OHCs), were simultaneously analyzed using hybrid solid-phase extraction (SPE) purification coupled to high-temperature gas chromatography-mass spectrometry (HTGC-MS). Methanol-based hybrid SPE increased the selective extraction, and HTGC resulted in a good chromatographic resolution for the separation of lipophilic compounds. The limits of quantification of cholesterol and CEs ranged from 0.2 to 10.0 μg/ml, while OHCs and cholesterol precursors ranged from 0.01 to 0.10 μg/ml. Linearity as the correlation coefficient was higher than 0.99 with the exception of cholesteryl laurate, myristate, oleate, and linoleate (r² > 0.98). The precision (% coefficient of variation) and accuracy (% bias) ranged from 1.1 to 9.8% and from 75.9 to 125.1%, respectively. The overall recoveries of CEs ranged from 26.1 to 64.0%, and the recoveries of other sterols ranged from 83.8 to 129.3%. The cholesterol signatures showed sex differences in patients with vasospastic angina and may associate with 24-reductases. This technique can be useful for making clinical diagnoses and for an increased understanding of the pathophysiology of vasospastic angina.     

Oral Glucose Tolerance and Related Factors in a Normal Population Sample—II. Interrelationship of Glycerides,Cholesterol, and other Factors with the Glucose and Insulin Response

The data available from the previous paper have been analysed to determine the interaction between the blood sugar and plasma insulin responses to oral glucose and a number of other biological variables. The total sugar and insulin responses were derived by calculating the total area and the incremental area under the curves.The blood sugar area was significantly correlated with age in both men and women, particularly the former. A striking degree of correlation was found in men between the level of fasting glycerides and the blood sugar area. There was a significant correlation also in women, but this was entirely due to the presence of the older, postmenopausal individuals. A lower degree of correlation was found in both sexes between fasting glycerides and the insulin area.Obesity, as defined by three interrelated factors—ponderal index, triceps fat-fold thickness, and arm girth —was significantly correlated with the insulin area (the association being stronger in the men) and with the blood sugar area only in men.The fasting cholesterol level was correlated with obesity in both sexes but with the blood sugar area and the insulin area only in men.     

Molecular mechanism of cyclodextrin mediated cholesterol extraction

The depletion of cholesterol from membranes, mediated by β-cyclodextrin (β-CD) is well known and documented, but the molecular details of this process are largely unknown. Using molecular dynamics simulations, we have been able to study the CD mediated extraction of cholesterol from model membranes, in particular from a pure cholesterol monolayer, at atomic resolution. Our results show that efficient cholesterol extraction depends on the structural distribution of the CDs on the surface of the monolayer. With a suitably oriented dimer, cholesterol is extracted spontaneously on a nanosecond time scale. Additional free energy calculations reveal that the CDs have a strong affinity to bind to the membrane surface, and, by doing so, destabilize the local packing of cholesterol molecules making their extraction favorable. Our results have implications for the interpretation of experimental measurements, and may help in the rational design of efficient CD based nano-carriers.