Erythrocyte Membrane Cholesterol Research Articles

Unmasking the morphological alteration of erythrocytes among women suffering from PCOS

Dyslipidemia is frequently observed in polycystic ovarian syndrome (PCOS). Changes in plasma lipid levels potentially alter erythrocyte membrane lipid composition due to lack of inbuilt lipid synthesis machinery. Therefore, development of morphologically altered erythrocytes in PCOS patients with dyslipidemia is expected. However, this has not been established so far. So, we took this opportunity to explore the morphological alterations among dyslipidemic PCO women. We recruited thirty-five dyslipidemic PCOS women (satisfying Rotterdam criteria, without medication) and twenty-five age-matched healthy controls. Scanning electron microscopy revealed a significant increase in the number of stomatocytes, acanthocytes, and echinocytes in the PCO group. PCO group showed a considerable decrease in plasma antioxidant levels. Elevated lipid peroxidation, protein carbonylation, and decreased free thiol group in erythrocyte membrane in PCOS suggest oxidative degradation of the erythrocyte membrane. Elevated intracellular ROS levels, increased methemoglobin formation, and a decrease in NADPH methemoglobin reductase in PCOS also indicate altered physicochemical property of hemoglobin due to oxidative overload. Additionally, these patients exhibit a rise in erythrocyte membrane cholesterol and triglyceride, which promotes the membrane to become less fluidic and less fragile. Thus, these results corroborate a potential role in altering erythrocyte morphology among dyslipidemic PCO women.

CSL112 Increases Plasma Cholesterol Efflux Capacity and Reduces Erythrocyte Membrane Cholesterol Content in Blood Samples from Patients with Sickle Cell Disease

Background: Lipid homeostasis is abnormal in sickle cell disease (SCD) and contributes to elevated erythrocyte cholesterol content which has been implicated in sickling. The highly oxidative environment in blood from patients with SCD alters high-density lipoprotein (HDL) composition and impairs HDL particle remodeling. Specifically, it negatively impacts on the capacity of HDL particles to mediate apolipoprotein A-I (apoA-I) exchange and protect against inflammation. Low apoA-I levels and altered composition of HDL have been well-documented at steady state, associated with high risk features of SCD, including endothelial dysfunction and risk of pulmonary hypertension. The low levels and activity of apoA-I fall even further during vaso-occlusive events in SCD. This diminished pool of apoA-I activity may mediate reduced capacity of HDL to extract cholesterol from erythrocyte membranes and increase risk of vascular dysfunction. Purpose: In this study we aimed to determine whether plasma derived apoA-I (CSL112; apolipoprotein A-I (human)) could elevate the cholesterol efflux capacity (CEC) of plasma from SCD patients and thereby reduce erythrocyte membrane cholesterol content. Methods: All SCD subjects (n=14) had confirmed HbSS genotype except one who was with HbS/β0-thalassemia, healthy control subjects (n=13) were not race-matched. CSL112 was incubated with fresh blood derived from control subjects and SCD patients for 6 hours at 37°C. The cholesterol content of ghost membranes of isolated erythrocytes was measured. HDL particle size distribution was assessed by native gel electrophoresis followed by western blotting. The ex vivo capacity of apoB-depleted plasma to efflux cholesterol was determined using [3H]cholesterol-loaded RAW264.7 macrophages. Results: CEC of SCD plasma was attenuated (75.9 ± 7.4%, n=14) compared to controls (100%, n=13), with a predominant decrease of ATP binding cassette transporter A1 (ABCA1)-dependent CEC (77.7 ± 9.2% vs 100%, respectively). Consistently, SCD plasma showed a trend towards lower levels of pre-β1 HDL (78.5 ± 6.5%) as compared to controls (100 %). Incubation with CSL112 raised plasma levels of small HDL species and pre-β1 HDL, resulting in a more than 3.5 fold increase of CEC in both patients and controls. Treatment of SCD blood with CSL112 reduced erythrocyte cholesterol content to a greater extent compared to control blood (% reduction: 12.8% ± 3.1% in SCD, 6.3% ± 5.4% in controls). Conclusion: Our data suggest that reduced levels of lipid-poor pre-β1 HDL in SCD plasma reflect impaired HDL particle remodeling and account for a decreased CEC. CSL112 may acutely generate HDL species which optimally support ABCA1-dependent cholesterol efflux and thereby improve HDL function, normalize erythrocyte membrane cholesterol content and correct the imbalance in cellular cholesterol homeostasis in SCD patients. ApoA-I as a potential therapeutic could have implications in the pathobiologic understanding and future clinical management of vaso-occlusive crisis and of chronic vascualopathy such as pulmonary hypertension.

The effect of selected bisphenols on model erythrocyte membranes of different cholesterol content

Bisphenols belong to the group of environmental pollutants with proven harmful impact on human red blood cells. However, the exact effect of these substances may vary depending on the lipid composition of the cell membrane, since this structure is the first barrier between the cell interior and the external environment. The aim of this work was to analyze the influence of bisphenol A (BPA), bisphenol S (BPS) and their 1:1 mixture on model human erythrocyte membranes, composed of sphingomyelin (SM), phospatidylcholine (PC) and cholesterol (Chol). Due to the postulated correlation between the content of cholesterol in biomembranes and the toxic effect of bisphenols the model systems of different sterol concentrations (10, 20 and 40 mol% of Chol) were used in the studies. In the experiments, Langmuir monolayer technique accompanied with Brewster Angle Microscopy were applied and liposome properties were investigated. The obtained findings reveal that, in the investigated range of the sterol content, the effect of BPA, namely the changes of the organization and stability of model membranes and weakening of the attractive lipid-lipid interactions, is strongly dependent on the concentration of Chol in the system. The higher the sterol content, the stronger the BPA-induced alterations in membrane properties. However taking into account the results reported previously for the system containing 33.3% of cholesterol, it seems that the relationship between the effect of BPA and the amount of Chol is not linear for higher sterol concentrations. In contrast, BPS shows a much weaker influence on model erythrocyte membranes and does not act selectively on the systems studied. The effect of a mixture of BPA and BPS is intermediate between that of BPA and BPS used separately, however, the observed effects appear to be determined only by the presence of BPA in the system. Thus, the concentration of cholesterol in human erythrocyte membranes, which depends on factors such as age or health status, may play a key role in the toxic effects of BPA but not BPS.

Dramatic Consequences of Reducing Erythrocyte Membrane Cholesterol on Plasmodium falciparum.

ABSTRACTCholesterol is the most abundant lipid in the erythrocyte. During its blood-stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in the intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-β-cyclodextrin (MβCD) was shown to inhibit parasite invasion and growth. In addition, MβCD treatment of trophozoite-stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that MβCD treatment for just 30 min causes dramatic expulsion of the trophozoite-stage parasites. This forceful expulsion occurs within 10 s. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented MβCD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of the infected erythrocyte plasma membrane.IMPORTANCE Malaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBCs), leading to their destruction. The parasite depends upon its host for much of its nutritional needs. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malaria parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here, we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in the physiology of malaria parasites.

Induced alteration of rat erythrocyte membrane with effect of pyrethroid based compounds.

The effects of tetramethrin and prallethrin exposure on plasma total proteins, free amino acids, albumins, urea, urea nitrogen, uric acid, creatinine were tested. Serum SGOT, SGPT and lipid profile, antioxidants super oxide dismutase (SOD), catalase, GSH, G-Px, phospholipids, cholesterol, C/P ratio in membranes of erythrocyte and membrane fluidity were analyzed. The reason of the study were analyzed to examine the possessions of mosquito repellent pyrethroid (MRP) based compounds tetramethrin and prallethrin exposure on plasma profile, antioxidant status of erythrocyte membrane, membrane fluidity in male Wistar rats. We tested chronically for three months exposure every day (continuously for 8–10 h per day by inhalation) of tetramethrin and prallethrin markedly available (MRP) repellents treated on male Wistar rats. Our results confirmed that tetrarmethrin and prallethrin treatment effect of plasma profile alterations, and lipid homeostasis mechanism in Red Blood cells (RBCs). Tetramethrin and prallethrin treatment significantly increased in erythrocyte membrane phospholipids and decreased levels of cholesterol with no change of protein content, increased C/P ration levels. Inhalation of tetramethrin and prallethrin stimulate plasma biophysical and biochemical modify SGOT, SGPT, erythrocyte membrane cholesterol and phospholipid levels, individual phospholipids and membrane fluidity of exposure rats compared to controls.

Cholesterol-dependent enrichment of understudied erythrocytic stages of human Plasmodium parasites

For intracellular pathogens, the host cell provides needed protection and nutrients. A major challenge of intracellular parasite research is collection of high parasite numbers separated from host contamination. This situation is exemplified by the malaria parasite, which spends a substantial part of its life cycle inside erythrocytes as rings, trophozoites, and schizonts, before egress and reinvasion. Erythrocytic Plasmodium parasite forms refractory to enrichment remain understudied due to high host contamination relative to low parasite numbers. Here, we present a method for separating all stages of Plasmodium-infected erythrocytes through lysis and removal of uninfected erythrocytes. The Streptolysin O-Percoll (SLOPE) method is effective on previously inaccessible forms, including circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. SLOPE can be used on multiple parasite species, under multiple media formulations, and lacks measurable impacts on parasite viability. We demonstrate erythrocyte membrane cholesterol levels modulate the preferential lysis of uninfected host cells by SLO, and therefore modulate the effectiveness of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples. Due to consumption of cholesterol by other intracellular bacteria and protozoa, SLOPE holds potential for improving research on organisms beyond Plasmodium.

Assessment of Membrane Erythrocyte Cholesterol Level in Sickle Cell Disease

Objective: The aim of this study was to assess the level of erythrocyte membrane cholesterol in sickle cell patients, which is one of the essential parameters of membrane fluidity that contributes to understanding the hemolytic state of the erythrocyte. Methods: We worked with blood specimens from 20 controls and 50 sickle cell patients. The blood count and the isoelectric focusing (IEF) were performed on the samples in order to select them. The titration of the erythrocyte membrane cholesterol was made after washing and lysing the erythrocytes with the hemolyzing solution (EDTA, 2 Mercapto-Ethanol, NADP, NaOH). The cholesterol level was assessed by the enzymatic colorimetric method. The results were analyzed by Student’s test. Results: We worked with 16 control subjects with a hemoglobin status and a normal hemogram. The evaluation of the erythrocyte membrane cholesterol level of the samples allows us to define the reference interval (α = 0.05) at 17.55 ± 3.83 mg of cholesterol/g of hemoglobin. The erythrocyte membrane cholesterol levels of sickle cell patients found in this study were 11.58 ± 2.98 mg cholesterol/g of hemoglobin. In this study, 38 sickle cell patients (76%) were found with a low erythrocyte membrane cholesterol level compared to the reference interval. Statistical analysis showed that there was a significant difference (α = 5%) between the erythrocyte membrane cholesterol level of normal subjects and sickle cell patients. Conclusion: Most of the sickle cells patients had a decreased erythrocyte membrane cholesterol level. This reduces the membrane fluidity making the erythrocyte membrane rigid and more fragile. This may be one of the factors responsible for the lyses of erythrocytes.

Red blood cell membrane cholesterol in type 2 diabetes mellitus

Type II diabetes mellitus (T2DM) affects an estimate of 450 million individuals. Hence, there remains an urgent need to explore the use of novel biomarkers with the aim of preventing and managing cardiovascular risk among these individuals. Hallmarks of this condition are lipid and glucose dyshomeostasis which are accompanied by a prothrombotic phenotype; these pose as eminent links between T2DM and cardiovascular disease. Diabetic dyslipidemia affects not only plasma lipid profiles but extends further into the haematological system, modulating the cholesterol concentration of erythrocyte membranes. Elevations in this biomarker implicate cell biophysics and contribute to an increased cardiovascular risk. Here we investigate the variation of membrane cholesterol levels in RBCs, as well as the degree of hypercoagubility between healthy and type II diabetic subjects. Furthermore, we provide an adapted method to assess erythrocyte membrane cholesterol levels using a quantitative and qualitative approach. In conclusion, individuals with type II diabetes exhibit elevated erythrocyte membrane cholesterol levels which may act as a prominent link between the diabetic state and subsequent cardiovascular complications. Ultimately, this erythrocyte parameter exhibits applicative biomarker potential and may provide clinical utility in terms of disease monitoring and prognostics.

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

Influence of Plasma Cholesterol on Erythrocyte Membrane Cholesterol in Type 2 Diabetic Subjects

Influence of Plasma Cholesterol on Erythrocyte Membrane Cholesterol in Type 2 Diabetic Subjects

The role of red blood cell deformability and Na,K-ATPase function in selected risk factors of cardiovascular diseases in humans: focus on hypertension, diabetes mellitus and hypercholesterolemia.

Deformability of red blood cells (RBC) is the ability of RBC to change their shape in order to pass through narrow capillaries in circulation. Deterioration in deformability of RBC contributes to alterations in microcirculatory blood flow and delivery of oxygen to tissues. Several factors are responsible for maintenance of RBC deformability. One of them is the Na,K-ATPase known as crucial enzyme in maintenance of intracellular ionic homeostasis affecting thus regulation of cellular volume and consequently RBC deformability. Decreased deformability of RBC has been found to be the marker of adverse outcomes in cardiovascular diseases (CVD) and the presence of cardiovascular risk factors influences rheological properties of the blood. This review summarizes knowledge concerning the RBC deformability in connection with selected risk factors of CVD, including hypertension, hyperlipidemia, and diabetes mellitus, based exclusively on papers from human studies. We attempted to provide an update on important issues regarding the role of Na,K-ATPase in RBC deformability. In patients suffering from hypertension as well as diabetes mellitus the Na,K-ATPase appears to be responsible for the changes leading to alterations in RBC deformability. The triggering factor for changes of RBC deformability during hypercholesterolemia seems to be the increased content of cholesterol in erythrocyte membranes.

Simvastatin-loaded nanostructured lipid carriers attenuate the atherogenic risk of erythrocytes in hyperlipidemic rats

This study aimed to investigate the effect of simvastatin (SV) loaded nanostructured lipid carriers (SV loaded NLCs) on atherogenic index (AI), erythrocytes membrane lipid and antioxidant/pro-oxidant status in hyperlipidemic rats. SV loaded NLCs were successfully prepared with desired nano-particles size, spherical shape, high encapsulation efficiency (EE %) and sustained SV release. The results of biological studies revealed that administration of SV loaded NLCs to rats increased SV bioavailability compared to SV suspension. Intraperitoneal injection of tyloxapol as hyperlipidemic agent induces a significant increase of plasma AI, uric acid, lipid peroxidation and protein oxidation. While, plasma total antioxidant capacity and paraoxonase-1 activity were significantly decreased. Moreover, tyloxapol induced-hyperlipidemia increases erythrocyte's membrane cholesterol and deteriorates erythrocyte's antioxidant enzyme activity, GSH/GSSG ratio and NO level However, the propagation of erythrocyte's pro-oxidant activity and hemolysis was observed. On the contrast, the treatment of these rats with SV loaded NLCs improved the measured parameters compared to rats received SV suspension and hyperlipidemic rats. The predominant effect of SV loaded NLCs may be attributed to the enhancement of absorption, prolonged duration and improvement of bioavailability of SV. Accordingly, SV loaded NLCs showed advantageous effects on the blood lipid levels and atherogenic risk of erythrocytes in hyperlipidemic conditions compared to SV suspension.

The Effect of Combined Ezetimibe/Atorvastatin Therapy vs. Atorvastatin Monotherapy on the Erythrocyte Membrane Structure in Patients with Coronary Artery Disease: A Pilot Study.

Erythrocytes play an important role in atherogenesis. An excessive accumulation of cholesterol in erythrocyte membranes leads to disruption of the erythrocytes. The aim of the study was to compare the effect of two different hypolipidemic therapies on the structure of erythrocyte membranes. The study included 18 patients with angiographic confirmed coronary artery disease who, despite at least 6 months of hypolipidemic treatment, had not achieved LDL-C < 70 mg/dL and 18 healthy individuals as the control group. The following parameters were studied: total cholesterol level and erythrocyte membrane fluidity, lipid peroxidation, SH groups in membrane protein and plasma lipids. We observed a decrease in TC (20%), LDL-C (35%), level of lipid peroxidation (25%) and total cholesterol in erythrocytes (23%), and an increase in HDL-C (8%) and erythrocyte membrane fluidity of subsurface layers (14%) after 6 months of 10 mg atorvastatin + 10 mg ezetimibe therapy, in comparison with healthy controls. In the group treated with 40 mg atorvastatin for 6 months, decreased LDL-C (23%), lipid peroxidation (37%) and membrane cholesterol concentration (18%) was noted, as well as an increase in erythrocyte membrane fluidity in the subsurface layers (12%). Both the combination therapy and the monotherapy lead to an improvement of erythrocyte membrane structure, whose parameters reached values close to those in the control healthy group.

Effects of Pistacia Atlantica Extract on Erythrocyte Membrane Rigidity, Oxidative Stress, and Hepatotoxicity Induced by CCl4 in Rats.

Background:Previous findings have suggested that antioxidants may reduce the levels of free radicals, which induce oxidative damage and play a key role in various diseases. Thus, we evaluated the protective activity of a Pistacia atlantica extract on erythrocyte membrane rigidity, oxidative stress, and hepatotoxicity induced by carbon tetrachloride (CCl4) in rats.Materials and Methods:Fresh leaves of P. atlantica were collected from the mountains in Yasuj, Iran. Acute oral toxicity (LD50) was evaluated in Wistar rats (200–230 g). Animals were randomly divided into 4 groups, out of which the negative and plant control groups received distilled water and P. atlantica extracts (500 mg/kg), respectively. The toxic rat group received CCl4, while the treatment group received CCl4 + P. atlantica extract. Blood plasma was utilized for the estimation of enzyme markers and lipid peroxidation, whereas hemolysate was applied for the determination of superoxide dismutase (SOD) and catalase activities. The levels of cholesterol and phospholipids in erythrocyte membranes were also determined. Rats were killed under anesthesia by cervical dislocation; liver was isolated from each rat and tissues homogenization was prepared for biochemical parameters such as malondialdehyde (MDA) and reduced glutathione (GSH) levels.Results:LD50 values were determined for doses >3000 mg/kg (p.o.). The activities of glutamic pyruvate transaminase (GPT), glutamic oxaloacetate transaminase (GOT), alkaline phosphatase (ALP) and GSH in the protected group were significantly (p < 0.001) reduced compared with those of toxic rats. In addition, we observed a decrease in the cholesterol level and an increase in red blood cell membrane phospholipids, SOD, and catalase activities (p < 0.001) in the protected group, as compared with toxic rats. Administration of Pistacia atlantica extract normalized liver tissue MDA level (p < 0. 01) when compared to CCl4 treated group.Conclusion:The P. atlantica extract was able to normalize the levels of biochemical markers, including liver enzyme markers, first-line defense enzymes, and lipid peroxidation markers.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin

Abstract Background Lipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. Methods and results Rabbits fed with atherogenic diet (0.75% cholesterol) for 5months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39±0.03mm 2 ), and lipid core size was associated with the concentration–time integral (CTI) of CEM levels ( r =0.567, P =0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3months, followed by either normal diet or normal diet plus rosuvastatin for the next 3months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. Conclusions Increased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.

Determination of cholesterol in erythrocyte membranes by HPLC method and its clinical application

Objective To develop a high performance liquid chromatographic method (HPLC) for the analysis of of cholesterol in erythrocyte membranes. Methods The study included 167 consecutive chest pain patients who underwent coronary artery angiography in the Department of Cardiology, Nanjing General Hospital of Nanjing Command between September 2012 and February 2013. According to the clinical symptoms and t angiographic results，patients were divided into three groups: acute coronary syndrome (ACS) group（n=46），stable angina pectoris (SAP) group （n=76）and the control group（n=45）. After the erythrocyte sample was hypotonically lysed and washed, saponification was carried out in a polassium hydroxide solution at 70 ℃. After extraction by Hexane/isopropanol mixture, the sample was separated on a Lichrospher column and detected by ultraviolet absorbance at 208 nm. A mobile phase composed of acetonitrile-isopropyl alcohol was found to be the most suitable for this separation. Concentrations of cholesterol in erythrocyte membranes were tested. Analysis of variance with covariates (ANOVA) was used to evaluate differences in CEM levels among groups. The relationship between continuous variables was evaluated by Spearman′s correlation coefficient. Results Under the chromatographic conditions described, retention time of the cholesterol was approximately 6.1 min. Good separation and detectability of cholesterol in erythrocyte membranes were obtained. The method proved to be linear in the injection range of cholesterol from 0.05 g to 2.00 g. Cholesterol content in erythrocyte membranes were (87.0 μg/mg, 75.4-98.9 μg/mg), (92.9 μg/mg, 83.8-109.0 μg/mg) and (173.9 μg/mg, 140.0-188.8 μg/mg) in the control, SAP and ACS groups, respectively. Cholesterol content in erythrocyte membranes was significantly higher in ACS group than that in SAP and control groups (P<0.01). Conclusion We have successfully developed a method for the determination of cholesterol in erythrocyte membranes with good sensitivity, specificity and repeatability. （Chin J Lab Med，2014,37：179-183） Key words: Erythrocyte membrane; Cholesterol; Chromatography, high pressure liquid; Acute coronary syndrome; Atherosclerosis

Erratum to: Cholesterol Forms and Traditional Lipid Profile for Projection of Atherogenic Dyslipidemia: Lipoprotein Subfractions and Erythrocyte Membrane Cholesterol

Erratum to: Cholesterol Forms and Traditional Lipid Profile for Projection of Atherogenic Dyslipidemia: Lipoprotein Subfractions and Erythrocyte Membrane Cholesterol

Cholesterol Forms and Traditional Lipid Profile for Projection of Atherogenic Dyslipidemia: Lipoprotein Subfractions and Erythrocyte Membrane Cholesterol

Atherogenic dyslipidemia characterized by abnormal changes in plasma lipid profile such as low high-density lipoprotein (HDL) and increased triglyceride (TG) levels is strongly associated with atherosclerotic diseases. We aimed to evaluate the levels of pro- and antiatherogenic lipids and erythrocyte membrane cholesterol (EMC) content in normo- and dyslipidemic subjects to investigate whether EMC content could be a useful marker for clinical presentation of atherogenic dyslipidemia. Low-density lipoprotein (LDL), HDL and their subfraction levels and erythrocyte lipid content were determined in 64 normolipidemic (NLs), 42 hypercholesterolemic (HCs) and 42 mixed-type dyslipidemic subjects (MTDs). Plasma atherogenic lipid indices [small–dense LDL (sdLDL)/less-dense HDL (LHDL), TC/HDL-C, TG/HDL-C and Apo B/AI] were higher in MTDs compared to NLs (p < 0.001). The highest sdLDL level was observed in HCs (p < 0.01). Despite a slight increase in EMC level in dyslipidemic subgroups, the difference was not statistically significant. A significant negative correlation, however, was observed between EMC and sdLDL/LHDL in HCs (p < 0.035, r = −0.386). Receiver operating characteristic curves to predict sdLDL level showed that TG and EMC levels had higher area under curve values compared to other parameters in HCs. We showed that diameters of larger LDL and HDL particles tend to shift toward smaller values in MTDs. Our results suggest that EMC content and TG levels may be a useful predictor for sdLDL level in hypercholesterolemic patients.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: Modulatory effects of rosuvastatin

BackgroundLipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. Methods and resultsRabbits fed with atherogenic diet (0.75% cholesterol) for 5months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39±0.03mm2), and lipid core size was associated with the concentration–time integral (CTI) of CEM levels (r=0.567, P=0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3months, followed by either normal diet or normal diet plus rosuvastatin for the next 3months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. ConclusionsIncreased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.

The Effects of Atorvastatin Therapy on Rheological Characteristics of Erythrocyte Membrane, Serum Lipid Profile and Oxidative Status in Patients with Dyslipidemia

The statins, most commonly used in the treatment of hyperlipidemia, have certain beneficial effects including improved endothelial function, plaque stability and decreased oxidative stress and inflammation, beyond their lipid-lowering effect in plasma. We evaluated the pleiotropic impact of atorvastatin on erythrocyte structural/mechanical properties and lipid peroxidation in dyslipidemics. The study group included 44 patients with dyslipidemia and was divided into subgroups according to triglyceride and cholesterol levels as hypercholesterolemic (n=29) and mixed-type hyperlipidemic (n=15). Subjects were given 10mg atorvastatin per day for 12weeks. Changes in serum lipid composition, lipid contents, Na(+)/K(+)-ATPase activity and osmotic fragility in erythrocytes and oxidative stress parameters of erythrocytes and plasma were studied. Atorvastatin therapy improved the serum lipid profile of both subgroups. This alteration was accompanied by a decreased level of cholesterol in erythrocyte membranes. Moreover, enhanced activity of Na(+)/K(+)-ATPase in erythrocytes reflected the improvements in membrane lipids of both subgroups. However, a significant change was observed in osmotic fragility values of the mixed-typed dyslipidemic group. This treatment lowered the lipid peroxidation in plasma and erythrocytes and increased plasma total antioxidant capacity in all groups. The present study shows that the use of atorvastatin reversed the structural and functional features of erythrocyte membranes in dyslipidemic subjects. Also, hypolipidemic therapy had a beneficial impact on a balance between oxidant and antioxidant systems.