Effect of a taurine treatment on the regression of existing atherosclerotic lesions in rabbits fed on a high-cholesterol diet

We studied whether taurine has any regressive effect on existing atherosclerotic lesions and lipid peroxidation in rabbits fed on a high-cholesterol (HC) diet. The cholesterol, triglyceride, malondialdehyde (MDA) and diene conjugate (DC) levels, as well as the aortic histopathological findings were examined in rabbits that had been fed on a cholesterol-containing diet for 8 months [0.5% cholesterol (w/w) for 3 months and subsequently 0.25% cholesterol (w/w) for 5 months], and then for a further 4 months on a normal diet with or without taurine treatment [1% (w/v) in the drinking water]. High levels of lipid and lipid peroxide induced by the HC diet were observed to decline in the plasma, liver and aorta of atherosclerotic rabbits, as well as a slight retardation in aortic atherosclerotic lesions during the regression period. Although no significant differences in the lipid and lipid peroxide levels in the plasma and aorta were found between the regressed groups with or without the taurine treatment, the extent of atherosclerotic lesions in the aorta was less in the taurine-treated regressed group than in the non-treated regressed group. However, the liver MDA and DC levels were lower in the regressed rabbits with the taurine treatment in the non-treated group. These results indicate that the taurine treatment may accelerate the regression of cholesterol-induced atherosclerotic lesions in rabbits without having any effect on the plasma and aorta lipid and lipid peroxide levels.     

Simultaneous occurrence of hypercholesterolemia and hemolytic anemia in rats fed cholesterol diet

Cholesterol diet-induced hemolytic anemia in rats was described. When rats were fed a cholesterol diet for 11 weeks, serum cholesterol rapidly increased within the first week, and was maintained in 5-10 times higher levels throughout the study as compared to those of control rats. Erythrocyte count, hematocrit and hemoglobin concentration decreased from about 2 weeks of feeding. The spleen showed an increase of hemosiderin deposition from 6 weeks of feeding. The half life of erythrocytes labelled with 51Cr was shortened significantly at 6 weeks of feeding. These findings indicate that cholesterol diet can induce hemolytic anemia. Serum cholesterol and phospholipid were markedly increased, but in erythrocyte membrane, free cholesterol content was not persistently increased and phospholipid content was decreased. In hemorrheological studies, erythrocyte deformability and mechanical hemolysis tended to reduce. In conclusion, it was considered that as a result of reduced phospholipid content the erythrocytes of cholesterol-fed rats were decreased in its deformability and were captured more easily by the spleen. The profile of hemolytic anemia in cholesterol-fed rats was quite different from those reported in cholesterol-fed guinea pigs, rabbits and dogs.     

The effect of thymosin fraction 5 on lipid peroxidation in rabbits fed a high-cholesterol diet

Plasma and erythrocyte lipid levels and susceptibility of erythrocytes to lipid peroxidation were determined in rabbits fed diet containing 2% (w/w) cholesterol, for 3 months. Hypercholesterolemic rabbits had high plasma and erythrocyte lipid peroxide levels as compared to control rabbits. After high-cholesterol diet, the rabbits in the experimental group were divided into two groups. The first group was fed a normal diet for 21 days and the second group was given normal diet plus thymosin F5 injections every other day for the same period. At the end of this period, plasma and erythrocyte lipid peroxide levels were significantly decreased in the group injected with thymosin F5.     

Improving effect of dietary taurine supplementation on the oxidative stress and lipid levels in the plasma,liver and aorta of rabbits fed on a high-cholesterol diet

The effect of a high-cholesterol diet with or without taurine on lipids and oxidative stress in the plasma, liver and aorta of rabbits was investigated. The animals were maintained on a basal diet (control), a high-cholesterol diet (HC, 1% w/w), or a high- cholesterol diet supplemented with taurine (HCHT, 2.5% w/w) for two months. Taurine has an ameliorating effect on atherosclerosis together with a decreasing effect on the cholesterol and triglyceride levels in rabbits fed on an HC diet. The HCHT diet caused a significant decrease in the malondialdehyde (MDA) and diene conjugate (DC) levels in the plasma, liver and aorta of rabbits as compared to the HC group. This treatment did not alter the antioxidant system in the liver of rabbits in the HC group. Our findings indicate that taurine ameliorated oxidative stress and cholesterol accumulation in the aorta of rabbits fed on the HC diet and that this effect may be related to its antioxidative potential as well as its reducing effect on serum lipids.     

Hazelnut oil administration reduces aortic cholesterol accumulation and lipid peroxides in the plasma, liver, and aorta of rabbits fed a high-cholesterol diet

Hazelnut oil (HO) is rich in monounsaturated fatty acids and antioxidants. We wanted to investigate the effect of HO on lipid levels and prooxidant-antioxidant status in rabbits fed a high-cholesterol (HC) diet. An HC diet caused significant increases in lipids and lipid peroxide levels in the plasma, liver, and aorta together with histopathological atherosclerotic changes in the aorta. Glutathione levels, glutathione peroxidase, and glutathione transferase activities decreased significantly, but superoxide dismutase activity and vitamin E and C levels remained unchanged in the livers of rabbits following HC diet. HO supplementation reduced plasma, liver, and aorta lipid peroxide levels and aorta cholesterol levels together with amelioration in atherosclerotic lesions in the aortas of rabbits fed an HC diet, without any decreasing effect on cholesterol levels in the plasma or liver. HO did not alter the antioxidant system in the liver in the HC group. Our findings indicate that HO reduced oxidative stress and cholesterol accumulation in the aortas of rabbits fed an HC diet.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

Cholesterol-rich diets have different effects on lipid peroxidation, cholesterol oxides, and antioxidant enzymes in rats and rabbits

The objective of this study was to compare the effect of cholesterol feeding of rats and rabbits. The levels of lipid peroxidation products and oxysterols in the plasma of the two species plus the antioxidant enzyme activities in the liver and erythrocytes were measured to explain their different susceptibilities to atherosclerosis. Our study showed that rats are less susceptible than are rabbits to the atherogenic effect of a cholesterol-rich diet because of differences in lipid peroxidation products as well as antioxidant enzymes activities in their livers. In rabbits, cholesterol feeding produced severe hypercholesterolemia (43-fold increase) and increased plasma and liver lipid peroxidation. Total as well as the individual oxysterol contents of 7alpha-, 7beta-hydroxycholesterol, alpha-epoxy, beta-epoxycholesterol, cholestanetriol, 7-keto, and 27-hydroxycholesterol significantly increased in the plasma of hypercholesterolemic (HC) rabbits. Erythrocyte glutathione peroxidase (GSH-Px) activity significantly decreased whereas catalase activity significantly increased in HC rabbits. In rats cholesterol feeding increased the plasma cholesterol only twofold and had no effect on plasma or liver lipid peroxidation. Only 7alpha- and 7beta-hydroxycholesterol increased and no change was observed in any of the antioxidant enzymes activity in the erythrocytes. Although cholesterol feeding caused a 10-fold increase of liver cholesterol as ester in both rats and rabbits, the antioxidant enzyme GSH-Px and catalase activities in the liver significantly increased in rats but significantly decreased in rabbits. The increase of GSH-Px and catalase activities in the liver of cholesterol fed rats could have a protective role against oxidation, thus preventing the formation of lipid peroxidation and oxysterols.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Effect of Cholesterol Content on Gramicidin S-Induced Hemolysis of Erythrocytes

Gramicidin S (GS) is a cyclo-decapeptide antibiotic with wide Gram+ and Gram− antimicrobial spectrum. However, its therapeutic application is very limited due to hemolytic activity of GS. The presence of cholesterol defines one of the most significant differences between eukaryotic plasma membranes and bacterial inner membranes. To find out the cholesterol effect on the GS hemolytic efficiency we compared GS-induced hemolysis of erythrocytes extracted from the blood of healthy donors against donors with atherosclerosis, “naturally” enriched with cholesterol. Our results show that increased cholesterol levels significantly attenuates yet does not abolishes the GS hemolytic activity. High levels of cholesterol content in erythrocyte membranes results in a decrease in the membrane fluidity and deformability leading to a decrease in the rate of GS interaction with membranes. The results obtained confirm that hydrophobic as well as electrostatic interactions must be involved in the binding of GS to cell membranes. Lipid peroxidation occurring within atherosclerotic erythrocytes leads to considerable decrease in the degree of GS-induced erythrocyte hemolysis in vitro. These results can be applied to the rational design of GS analogs with increased antibacterial efficiency but reduced hemolytic activity.     

Effect of retinol on the hemolysis and lipid peroxidation in vitamin E deficiency

A study on the effect of retinolin vitro on the hemolysis of vitamin E deficient rat red blood cells showed that retinol enhanced the lysis of the E deficient cells as compared to the lysis of normal cells. The lipid peroxidation present during hydrogen peroxide induced lysis of E deficient cells was however markedly inhibited in the presence of retinol without affecting the rate of lysis. In an actively peroxidising system of non-enzymatic lipid peroxidation of rat liver or brain homogenates and of brain lysosomes incubated with human erythrocytes, no lysis was obtained; incorporation of retinol in such systems resulted in lysis but no peroxidation. Hydrogen peroxide generating substances almost completely inhibited the lysis of normal human erythrocytes by retinol, but linoleic acid hydroperoxide and auto-oxidised liver or brain homogenates and ox-brain liposomes increased the lysis. It is concluded that vitamin E deficient erythrocyte hemolysis may be augmented by retinol, an anti-oxidant, having a lytic function without the peroxidation of stromal lipids     

Role of the nonspecific modification of erythrocyte membrane lipids during hibernation in the suslik Citellus parryi

Studies have been made on the content of cholesterol, phospholipids, fatty acid composition, the intensity of lipid peroxidation, the activity of Na+, K+-ATPase, as well as on the peroxide hemolysis in the erythrocytes in prehibernating and hibernating ground squirrels. Changes in partial content of cholesterol and in fatty acid composition of membranes are presumably due to the excessive lipid peroxidation during hibernation resulting from the decrease in the activity of antioxidative enzymes, which also accounts for the increase in peroxide hemolysis of erythrocytes.     

Peroxyl Radical-Mediated Hemolysis: Role of Lipid, Protein and Sulfhydryl Oxidation

The objective of this study was to define the relationship between peroxyl radical-mediated cytotoxicity and lipid, protein and sulfhydryl oxidation using human erythrocytes as the target mammalian cell. We found that incubation of human erythrocytes with the peroxyl radical generator 2,2' azobis (2-amidinopropane) hydrochloride (AAPH) resulted in a time and dose-dependent increase in hemolysis such that at 50 mM AAPH maximum hemolysis was achieved at 120min. Hemolysis was inhibited by hypoxia and by the addition of certain water soluble free radical scavengers such as 5-aminosalicylic acid (5-ASA), 4-ASA, N-acetyl-5-ASA and dimethyl thiourea. Peroxyl radical-mediated hemolysis did not appear to involve significant peroxidation of erythrocyte lipids nor did they enhance protein oxidation at times preceding hemolysis. Peroxyl radicals did however, significantly reduce by approximately 80% the intracellular levels of GSH and inhibit by approximately 90% erythrocyte Ca²⁺ -Mg²⁺ ATPase activity at times preceding the hemolytic event. Our data as well as others suggest that extracellular oxidants promote the oxidation of intracellular compounds by interacting with certain redox active membrane components. Depletion of intracellular GSH stores using diamide did not result in hemolysis suggesting that oxidation of GSH alone does not promote hemolysis. Taken together, our data suggest that neither GSH oxidation, lipid peroxidation nor protein oxidation alone can account for peroxyl radical-mediated hemolysis. It remains to be determined whether free radical-mediated inactivation of Ca²⁺-Mg²⁺ ATPase is an important mechanism in this process.     

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane)

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios ≥1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Intralipid-infusion on erythrocyte membrane in rabbits

Through Intralipid infusion in rabbits, the phospholipids derived from Intralipid were incorporated into erythrocytes, although Intralipid is mainly composed of triglycerides. This is supported by the increase in oleic acid and the compensatory decrease in linoleic acid of the phospholipids in the erythrocyte membrane, corresponding to the content of linoleic acid in the phospholipids from Intralipid. The excess phospholipid rendered the membrane more fluid, probably by overwhelming the rigidifying effect of the increased cholesterol content. Furthermore, the shape of erythrocytes was changed from biconcave to spur, dose dependently. The morphological alterations in erythrocyte membranes could not be completely elucidated by the changes in lipid. These results suggested that the alteration in lipid metabolism in Intralipid-infused rabbits caused various effects on the erythrocyte membrane, through the elevation of triglyceride, cholesterol, and phospholipid contents in plasma.     

Influence of cholesterol and prostaglandin E1 on the molecular organization of phospholipids in the erythrocyte membrane. A fluorescent polarization study with lipid-specific probes

Anthryl-labeled fluorescent probes closely mimicking phosphatidylcholine and sphingomyelin were applied to study the state of these phospholipids in the rabbit erythrocyte membrane. At normal cholesterol levels both probes exhibited higher fluorescence polarization values in the membranes than in phospholipid vesicles of similar lipid composition, indicating a decreased fluidity of the probe environment in erythrocyte ghosts. In ghosts prepared from normal erythrocytes no evidence of lateral separation of phosphatidylcholine and sphingomyelin was found. At higher cholesterol levels, however, these lipids appear to segregate. Probably the effect of cholesterol on the erythrocyte membrane lipids involves lipid-protein interactions. At physiological concentrations, prostaglandin E1 only weakly affects the state of phosphatidylcholine and sphingomyelin in erythrocyte membranes. Cholesterol enrichment amplifies the effect of prostaglandin E1. Although the prostaglandin E1-induced changes depended much upon whether the ghosts were enriched with cholesterol in vitro or in vivo, with both types of ghosts effects of prostaglandin E1 were seen at extremely low effector concentrations that may have presented a few molecules of prostaglandin per ghost. The structural and functional significance of these findings is discussed.     

Interrelationship of antioxidative status, lipid peroxidation, and lipid profile in insulin-dependent and non-insulin-dependent diabetic patients

This study aimed to investigate the interrelationship of plasma lipid profile, lipid peroxidation, and erythrocyte antioxidative defense in patients with insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus. Plasma levels of total cholesterol, triglycerides, and lipid peroxides and the activities of copper, zinc superoxide dismutase (CuZnSOD), catalase, glutathione peroxidase (GSH-Px), as well as the amount of glutathione in erythrocytes, were determined in IDDM, NIDDM, and nondiabetic control subjects. Additionally, morphology of erythrocytes in all subjects was examined. Plasma levels of total cholesterol and triglycerides were significantly increased in NIDDM compared with controls. Also, the lipid peroxide level was higher in NIDDM than in either control or IDDM subjects. CuZnSOD activity in erythrocytes was elevated in NIDDM patients compared with the control. In NIDDM patients, more extensive erythrocyte spherocytosis and echinocytosis compared with both control and IDDM subjects were observed. In contrast with the IDDM group, the observed abnormality in lipid metabolism in NIDDM patients is closely associated with increased lipid peroxidation, changes in antioxidative defense, and erythrocyte morphology.     

Glycerol permeability of human fetal and adult erythrocytes and of a model membrane

When erythrocytes from different mammalian species are compared, the hemolysis rate in 0.3 m glycerol is seen to be directly related to the percentage of lecithin in the erythrocyte phospholipid. Since this percentage is higher in erythrocytes from human adults than in those from infants, the hemolysis times in 0.3 m glycerol were compared. As expected, hemolysis was more rapid in the adult cell, which is therefore more permeable to glycerol under these conditions. The permeability to glycerol of a film of erythrocyte lipids in vitro was next examined in a model system containing the two phases water and butanol. Lipid introduced into the bulk butanol appears as a film at the interface. When equal amounts of total lipid extracted from adult and fetal erythrocytes were introduced into the butanol phase of two such chambers, the initial flux of glycerol-(14)C across the lipid boundary was greater in the cell containing lipid from adult erythrocytes than in the cell containing fetal erythrocyte lipid. This difference corresponds qualitatively to the difference in hemolysis time measured in the intact erythrocytes.     

Peroxidation-induced perturbations of erythrocyte lipid organization

Peroxidation of erythrocyte membrane lipids by hydrogen peroxide perturbs the lipid bilayer and increases phagocytosis by macrophages. This study addresses the underlying mechanism of these processes, and in particular the role of malondialdehyde, a major byproduct of lipid peroxidation. When erythrocytes were treated with hydrogen peroxide or ascorbate/iron to generate malondialdehyde, or with malondialdehyde itself, only those cells treated with hydrogen peroxide showed increased phospholipid spacing and enhanced phagocytosis. This result indicates that the alterations observed are unique to hydrogen peroxide treatment, and that malondialdehyde does not play a role in inducing these changes in surface properties. Comparison of adherence to human umbilical vein endothelial cells and phagocytosis showed that increased phagocytosis was not mirrored by enhanced adherence. This result suggests that two different signals may mediate recognition of erythrocytes by macrophages and by endothelial cells.