Activation of human blood monocytes by oxidized polyunsaturated fatty acids: a possible mechanism for the generation of lipid peroxides in the circulation.

The ability of native and oxidized lipids and lipoproteins to stimulate production of reactive oxygen species (ROS; superoxide and hydrogen peroxide) by human blood monocytes has been studied in vitro. Neither native human low density lipoprotein (LDL), ''altered'' LDL (oxidized either by lipoxygenase, activated human monocytes or air) nor oxidized cholesterol had any significant effect on ROS production of monocytes. However, different oxidation products of a lipid emulsion (Lipofundin; largely consisting of linoleic acid oxidized either by lipoxygenase, Fe3+ or ultraviolet irradiation) greatly enhanced ROS production of monocytes. A hypothesis that activation of circulating leucocytes by oxidized fatty acids may generate oxidized plasma LDL, was tested in rabbits. Characteristics of LDL, separated from rabbit plasma 6 h after intravenous injection of an oxidized lipid emulsion, was compared to that of LDL isolated before the lipid treatment. Post-treatment LDL-fraction of plasma had increased lipid peroxide content and compared to the pretreatment LDL, caused a threefold increase in the incorporation of cholesterol into cultured (rat aortic) endothelial cells. The observed intense and lasting stimulation of monocytes by oxidized polyunsaturated fatty acids in vitro, and the generation of ''altered'' LDL by these oxidized lipids in vivo suggests a mechanism by which atherogenic oxidized LDL could form in the circulation.     

Effect of exercise training on in vitro LDL oxidation and free radical-induced hemolysis: the HERITAGE Family Study

Oxidant stress and overproduction of reactive oxygen species (ROS) contribute to the development of cardiovascular disease. Oxidative modifications of low-density lipoproteins (LDL) are thought to play an early and critical role in atherogenesis. LDL oxidation can be reproduced in vitro, but results usually show a large interindividual variation not entirely explained by the environment. Free radical-induced hemolysis is also proposed to reveal the overall antioxidant capacity. The roles of genetic factors and exercise on the variability of both measures were investigated. The study was conducted in 146 healthy individuals from 28 families participating in a 20-week exercise-training program. In addition to important biological and environmental influences on variation, significant familial aggregation was detected in all oxidation measures. Exercise did not significantly modify the LDL oxidation parameters, but significantly increased resistance was observed in the free radical-induced hemolysis, especially in women, this effect was not observed in smokers. In total, the findings suggest the presence of familial effects in the response to ex vivo oxidation. Further, smoking negates the beneficial effect of exercise training on erythrocyte resistance to free radical-induced hemolysis. These observations emphasize the importance of context in the evaluation of exercise and oxidant stress.     

Increased levels of low-density lipoprotein oxidation in patients with familial hypercholesterolemia and in end-stage renal disease patients on hemodialysis

Patients with familial hypercholesterolemia (FH) and patients with end-stage renal disease (ESRD) undergoing dialysis suffer from accelerated atherosclerosis. Oxidation of low-density lipoprotein (LDL) cholesterol is crucial in atherogenesis. In the present study, we determined the LDL oxidation level and oxidizability of isolated LDL of 11 male patients with FH, 15 male ESRD patients on hemodialysis, and 15 age-matched male normolipidemic healthy controls. FH patients were without lipid-lowering medication for at least 4 weeks and were reassessed after 2 years of cholesterol-lowering therapy (statins). LDL oxidation level was measured by ELISA using monoclonal antibody 4E6 to oxidized LDL (oxLDL) as the capture antibody and anti-human apoB antibody for detection; results were expressed as percentage oxLDL. In FH patients and in ESRD patients on hemodialysis, both groups having a higher percentage of cardiovascular disease, mean plasma LDL oxidation levels were significantly elevated compared with controls (4.9 +/- 1.3; 3.7 +/- 2.0; 1.7 +/- 0.6%, respectively). Within each group of subjects, LDL oxidation level was not associated with history of cardiovascular disease. Furthermore, in neither group was a significant correlation found between plasma concentration of LDL cholesterol and LDL oxidation level. After cholesterol-lowering therapy, LDL oxidation level in FH patients had not changed significantly and remained elevated compared with controls, despite a reduction of LDL cholesterol by 55% on average. Also, absolute plasma oxLDL concentrations, obtained by multiplying LDL oxidation level with plasma LDL cholesterol concentration, were significantly higher in FH patients before and after cholesterol-lowering therapy and in ESRD patients on hemodialysis than in controls (489 +/- 145; 189 +/- 122; 100 +/- 65; and 59 +/- 27 micro moles/L, respectively). No correlation was found between plasma oxLDL concentration and parameters of LDL oxidizability, LDL fatty acids, and LDL alpha-tocopherol content. We conclude that cholesterol-lowering therapy does not normalize elevated LDL oxidation levels in FH patients and elevated LDL oxidation level in FH and in ESRD might mirror atherosclerosis.     

溶血与非溶血时血浆及红细胞过氧化氢诱发化学发光值比较

本实验观察了血细胞对血浆过氧化氢诱发化学发光值（Ｈ２Ｏ２－ＩＣＬ）的影响。结果表明，正常兔血浆Ｈ２Ｏ２－ＩＣＬ值明显高于全血和红细胞悬液，发生溶血后上述三组份的发光值均显著增加，其中全血和血浆的发光值分别增加１５．５和６．１倍。油酸肺损伤兔所有组份的发光值均显著高于对照兔，同时其血浆Ｈ２Ｏ２－ＩＣＬ发光值的衰变系数明显低于对照兔。以上结果提示：红细胞及其碎片显著影响血浆Ｈ２Ｏ２－ＩＣＬ值，溶血促使     

氧化修饰和乙酰修饰低密度脂蛋白在A型清道夫受体基因敲除小鼠的清除

目的：阐明清道夫ＡＩ／Ｉ受体（ＳＲ－ＡＩ／Ｉ）在体内ＯＸ－ＬＤＬ和ＡＣ－ＬＤＬ清除中的作用和重要性。方法：观察同位素标记化学修饰ＬＤＬ在ＳＲ－ＡＩ／Ｉ基因敲除和正常小鼠血浆的清除率和同位素标记的ＬＤＬ在体内主要器官的分布。结果：ＯＸ－ＬＤＬ在正常小鼠血浆清除是非常快的,在５ｍｉｎ内,注入量的９０％即被清除。在ＳＲ－ＡＩ／Ｉ基因敲除小鼠,ＯＸ－ＬＤＬ在血浆的清除速率和正常小鼠相同。ＡＣ－ＬＤＬ的清除在对照组和ＳＲ－ＡＩ／Ｉ受体敲除小鼠也相似。同位素标记的ＡＣ－ＬＤＬ在血浆的清除能够完全被５０倍高剂量未标记的ＡＣ－ＬＤＬ阻断,但同样剂量的未标记的ＡＣ－ＬＤＬ仅能抑制ＯＸ－ＬＤＬ在血浆清除的５％。ＯＸ－ＬＤＬ和ＡＣ－ＬＤＬ主要被肝脏清除。组织器官放射性标记的ＯＸ－ＬＤＬ和ＡＣ－ＬＤＬ的分布在正常小鼠和ＳＲ－ＡＩ／Ｉ基因敲除小鼠没有差异。结论：ＳＲ－ＡＩ／Ｉ受体在体内ＯＸ－ＬＤＬ清除过程中不起主要作用。     

Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation?

PurposeMelatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation.Material/MethodsHuman erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37°C either in absence or presence of melatonin at concentration range 0.02 mM–3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO₂, GSH, fluorescent label DCFH₂) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis.ResultsThe prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO₂ and DCFH₂ was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%?h^?1) was 50% lower than that of the control while the HbO2 oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage.ConclusionsProbably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO2 and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.     

Susceptibility of plasma low- and high-density lipoproteins to oxidation in patients with severe hyperhomocysteinemia

 A moderate increase in plasma homocysteine is increasingly considered an important risk factor of atherosclerosis and thrombosis. However, the mechanisms by which hyperhomocysteinemia induces vascular disease are not well defined. In vitro studies suggest that cysteine and homocysteine can induce oxidative modification of low-density lipoproteins (LDL). This suggestion is relevant because lipoprotein oxidation is thought to play a key role in the development of atherosclerosis and in the triggering of thrombotic events. An attractive model to study this topic is provided by patients with classical homocystinuria, an inherited disease characterized by severe hyperhomocysteinemia and a high incidence of thromboembolisms. We investigated the existence of oxidized LDL and the susceptibility to oxidation of the plasma cholesterol-rich lipoproteins in six patients with severe hyperhomocysteinemia, most likely due to classical homocystinuria, and compared the results with matched controls. The proportion of electronegative LDL and the concentration of thiobarbituric acid reactive substances in native LDL and high-density lipoproteins (HDL) did not differ between patients and controls, suggesting that the proportion of modified lipoproteins is not increased in patients with severe hyperhomocysteinemia. The susceptibility to oxidative modification of plasma LDL and HDL was also similar in the two groups, although the patients had homocysteine levels 18.3-fold higher than controls. Thus, increased oxidative modification is not likely to be a relevant mechanism in explaining their high incidence of vascular disease. A possible explanation for the lack of increased susceptibility to oxidation, as would be expected for the metabolic blockade that causes classical homocystinuria, is the 4.1-fold decrease in the concentration of cysteine in the plasma of patients. As a result the total concentration of homocysteine plus cysteine was slighty lower in patients than in controls. This interpretation implies that more studies are needed on lipoprotein susceptibility to oxidation in patients in which both plasma homocysteine and cysteine concentrations are increased. This metabolic situation may be frequent in the population with moderate hyperhomocysteinemia and vascular disease. Received: 19 June 1996 / Accepted: 18 September 1996     

Oxidation-induced aggregation of LDL increases their uptake by smooth muscle cells from human aorta

Oxidative modification of human blood LDL induced by Cu²⁺, NaOCl, or 2,2-azobis-(2-aminopropane hydrochloride) was followed by their partial aggregation. Separation of oxidized LDL into aggregates and nonaggregated particles showed that they are characterized by a similar degree of oxidative modification. In contrast to nonaggregated particles, LDL aggregates in the same concentration significantly increased cholesterol content in smooth muscle cells from the intact (no involoved in atherosclerosis) human aortic intima. Our results indicate that atherogenicity of LDL oxidized by various factors is mainly associated with the formation of aggregates, but does not depend on the degree of oxidative modification. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 2, pp. 159–162, February, 2007     

Role of the kidneys in the mechanism of erythorodieresis in hemolytic (phenylhydrazine) and acute posthemorrhagic anemia

The degree of hemolysis was investigated in intact and nephrectomized rats after administration of phenylhydrazine. Hemolytic (phenylhydrazine) anemia in nephrectomized animals is manifested by a smaller decrease in the total erythrocyte count, the percentage of⁵¹Cr-labeled erythrocytes, and the intensity of the reaction for hemosiderin in tissues of the reticuloendothelial system. A lower degree of erythrodieresis was found in nephrectomized rats and after acute unreplaced blood loss. Perfusion of blood through the kidney of anemized rats leads to an increase in the plasma potassium concentration of the perfusion fluid, a decrease in the electrophoretic mobility and hemolysis time of the erythrocytes, an increase in their fragility, and a decrease in the proportion of protein fractions with molecular weights of between 74,500 and 27,000 in the erythrocyte stroma.Department of Pathological Physiology and General Chemistry, Altai Medical Institute, Barnaul. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1311–1313, November, 1976.     

Oxidative modifications of LDL increase its binding to extracellular matrix from human aortic intima: influence of lesion development, lipoprotein lipase and calcium

Retention of atherogenic lipoproteins in the arterial intima by extracellular matrix (ECM) is assumed to occur during early atherogenesis and its further development. Low density lipoprotein (LDL) trapped in the intima may undergo oxidative modifications, which initiate a chain reaction in atherogenesis. Lipoprotein lipase (LPL) has been found to mediate the binding of native and oxidized LDL to ECM produced by cultured cells and to contribute to foam cell formation by mildly oxidized LDL. In this study ECM, isolated from human aortic intima with different atherosclerotic lesions, was used for the first time to measure the binding to it in vitro of native and differently oxidized 125I-LDL. Oxidation of 125I-LDL increased its binding to the ECM, which was most prominent with the material isolated from intima at the early stage of atherogenesis. With the progression of atherosclerosis, the ability of the isolated intimal ECM to bind native and oxidized 125I-LDL decreased, and strongly oxidized 125I-LDL decreased more than native and moderately oxidized 125I-LDL. LPL increased the binding of moderately oxidized 125I-LDL to the ECM more than native 125I-LDL, while it had only a small effect on strongly oxidized 125I-LDL. LPL-mediated binding of native and oxidized 125I-LDL decreased with the development of atherosclerotic lesions. Calcium ions also increased the binding of LDL to the ECM. This enhanced binding increased with the extent of LDL oxidation, especially at the early stage of atherogenesis, and decreased with lesion progression. These data suggest that the ability of ECM to retain LDL in arterial intima depends on LDL oxidation status and changes with the progression of atherogenesis. In addition, LPL and calcium ions may participate in the retention of LDL in vivo.     

Effects of oxidized fibrinogen on the functions of blood cells,blood clotting,and rheology

Oxidatively-modified fibrinogen induces platelet aggregation and potentiates ADP-induced platelet aggregation and production of active oxygen forms in zymosan-stimulated leukocytes. Fibrinogen induces IL-8 production in primary culture of endothelial cells from human umbilical vein; the oxidized form of fibrinogen is more active, similarly as during induction of the expression cell adhesion molecules (P-selectin and ICAM-1). Oxidized fibrinogen (10 and 20% oxidation degree) impairs microrheological properties of the blood, sharply reduces erythrocyte deformability, modifies blood viscosity, and reduces suspension stability of the blood. Oxidized fibrinogen modified blood clotting parameters and ADP-, ristocetin-, and collagen-induced platelet aggregation in whole blood. Oxidized fibrinogen disordered the formation of fibrin clot and blood clotting process. Platelet aggregation was activated in response to ADP, but not to ristocetin and collagen, the degree of activation increased in direct proportion to the degree of fibrinogen oxidation. This indicates the “dysregulatory” effect of oxidized fibrinogen on platelets. The formation of platelet complexes with polymorphonuclear leukocytes was intensified in the presence of oxidized fibrinogen; polymorphonuclear leukocyte luminol-dependent fluorescence intensity in the presence of platelets increased after incubation with oxidized fibrinogen in comparison with native fibrinogen. Hence, oxidized fibrinogen plays an important role in the development of atherosclerosis and its complications (thromboses). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Supplement 2, pp. 21–32, April, 2007     

“In vitro” studies on the fate of diphenylhydantoin in uremia

“In vitro” studies were undertaken to evaluate the fate of DPH in blood of uremic patients.DPH incubated for 18 h at 37°C disappears from plasma in the presence of erythrocytes and more when the blood underwent hemolysis. This phenomenon was more pronounced in uremic blood.These findings suggest not only that DPH is absorbed onto the erythrocyte membrane, but also that it is bound by erythrocyte contents.It could be possible that, because of a low binding capacity of the albumin carrier in uremic blood, the drug penetrates more easily the erythrocyte membrane and is bound there. An intraery throcyte enzymatic metabolism of DPH is not excluded.     

Platelet interaction with bioactive lipids formed by mild oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) generates pro-inflammatory and pro-thrombotic mediators that play a crucial role in cardiovascular and inflammatory diseases. Mildly oxidized LDL (mox-LDL) and minimally modified LDL (mm-LDL) which escape the uptake of macrophage scavenger receptors accumulate in the atherosclerotic intima. Oxidatively modified LDL is also present within the electronegative LDL fraction in blood, which is elevated in patients at high risk for cardiovascular diseases. Mox-LDL and mm-LDL, but not native LDL are able to induce platelet shape change and aggregation. LDL oxidation generates lipids with platelet stimulatory properties such as lysophosphatidylcholine, certain oxidized phosphatidylcholine molecules, F(2)-isoprostanes and lysophosphatidic acid (LPA). Mox-LDL and mm-LDL are like a Trojan horse carrying these biologically active lipids and attacking cells through activation of physiological receptors and signaling mechanisms. LPA has been identified as the lipid responsible for platelet stimulation by mox-LDL, mm-LDL and also mox-HDL. These lipoproteins activate platelets by stimulating G-protein coupled LPA receptors and a Rho/Rho kinase signaling pathway leading to platelet shape change and subsequent aggregation. LPA-mediated platelet activation might contribute to arterial thrombus formation after rupture of atherosclerotic plaques and to the increased blood thrombogenicity of patients with cardiovascular diseases.     

Effect of Antioxidant Probucol on Cell-Mediated LDL Oxidation in Vitro and in Vivo

We studied the effect of phenol antioxidant probucol on free radical oxidation of LDL isolated from blood plasma of healthy donors. Oxidation was induced by co-incubation of LDL with cultured peripheral blood monocyte-macrophages and human umbilical vein endothelial cells under conditions of ischemia-reperfusion. In addition, the effect of probucol therapy on oxidability of plasma LDL in CHD patients was examined. Probucol (0.1-10 M) efficiently protected LDL from free radical oxidation in vitro and in vivo.     

高密度脂蛋白氧化修饰与动脉硬化

大量流行病学研究证实 ,血清高密度脂蛋白(highdensitylipoprotein ,HDL)水平与动脉粥样硬化(atherosclerosis ,AS)性心脑血管疾病的发病率呈负相关 ,HDL水平降低是不亚于低密度脂蛋白 (lowdensitylipoprotein ,LDL) ,甚至比LDL更显著的AS血管疾病的危险因素[1] 。动脉壁处LDL氧化是AS始动及发展的关键这一观点已得到充分肯定。近年来发现 ,在某些外在或内在的诱因下 ,作为AS保护因子的HDL也发生与LDL相似的氧化修饰 ,HDL氧化后 ,不仅理化性质发生明显变化 ,生物学功能也发生显著改变。本文就HDL氧化易感性、体内可能的氧化部…     

Lycopene synergistically inhibits LDL oxidation in combination with vitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic

Several lines of evidence suggest that oxidatively modified low-density lipoprotein (LDL) is atherogenic, and that atherosclerosis can be attenuated by natural antioxidants, which inhibit LDL oxidation. This study was conducted to determine the effect of tomato lycopene alone, or in combination with other natural antioxidants, on LDL oxidation. LDL (100 microg of protein/ml) was incubated with increasing concentrations of lycopene or of tomato oleoresin (lipid extract of tomatoes containing 6% lycopene, 0.1% beta-carotene, 1% vitamin E, and polyphenols), after which it was oxidized by the addition of 5 micromol/liter of CuSO4. Tomato oleoresin exhibited superior capacity to inhibit LDL oxidation in comparison to pure lycopene, by up to five-fold [97% vs. 22% inhibition of thiobarbituric acid reactive substances (TBARS) formation, and 93% vs. 27% inhibition of lipid peroxides formation, respectively]. Because tomato oleoresin also contains, in addition to lycopene, vitamin E, flavonoids, and phenolics, a possible cooperative interaction between lycopene and such natural antioxidants was studied. A combination of lycopene (5 micromol/liter) with vitamin E (alpha-tocopherol) in the concentration range of 1-10 micromol/liter resulted in an inhibition of copper ion-induced LDL oxidation that was significantly greater than the expected additive individual inhibitions. The synergistic antioxidative effect of lycopene with vitamin E was not shared by gamma-to-cotrienol. The polyphenols glabridin (derived from licorice), rosmarinic acid or carnosic acid (derived from rosemary), as well as garlic (which contains a mixture of natural antioxidants) inhibited LDL oxidation in a dose-dependent manner. When lycopene (5 micromol/liter) was added to LDL in combination with glabridin, rosmarinic acid, carnosic acid, or garlic, synergistic antioxidative effects were obtained against LDL oxidation induced either by copper ions or by the radical generator AAPH. Similar interactive effects seen with lycopene were also observed with beta-carotene, but, however, to a lesser extent of synergism. Because natural antioxidants exist in nature in combination, the in vivo relevance of lycopene in combination with other natural antioxidants was studied. Four healthy subjects were administered a fatty meal containing 30 mg of lycopene in the form of tomato oleoresin. The lycopene concentration in postprandial plasma was elevated by 70% in comparison to plasma obtained before meal consumption. Postprandial LDL isolated 5 hr after meal consumption exhibited a significant (p < 0.01) reduced susceptibility to oxidation by 21%. We conclude that lycopene acts synergistically, as an effective antioxidant against LDL oxidation, with several natural antioxidants such as vitamin E, the flavonoid glabridin, the phenolics rosmarinic acid and carnosic acid, and garlic. These observations suggest a superior antiatherogenic characteristic to a combination of different natural antioxidants over that of an individual one.     

总红细胞破损在滚柱泵体外转流实验中存在的研究

观察滚柱泵在长时间转注中是否存在“总红细胞破损”现象,用Ｐｏｌｙｓｔａｎ泵和Ｃｏｂｅ泵转流ｄＡＣＤ血４００ｍｌ,用联苯胺显示法测定游离血红蛋白,用Ｋｏｌｌｅｒ我法计算溶血指数,血样本为转流前,转中４、６、８、１０、１２、１４、１６ｈ。结果：两了血红蛋白随流时间延长,逐渐增高,两组溶血指数分别为０．２９６０ｍｇ／Ｌ和０．３９９３ｍｇ／Ｌ,两组比较无显著差异。在滚柱泵单体外转流实验中未观察到“总红细胞     

Influence of cocoa flavanols and procyanidins on free radical-induced human erythrocyte hemolysis

Cocoa can be a rich source of antioxidants including the flavan-3-ols, epicatechin and catechin, and their oligomers (procyanidins). While these flavonoids have been reported to reduce the rate of free radical-induced erythrocyte hemolysis in experimental animal models, little is known about their effect on human erythrocyte hemolysis. The major objective of this work was to study the effect of a flavonoid-rich cocoa beverage on the resistance of human erythrocytes to oxidative stress. A second objective was to assess the effects of select purified cocoa flavonoids, epicatechin, catechin, the procyanidin Dimer B2 and one of its major metabolites, 3'-O-methyl epicatechin, on free radical-induced erythrocyte hemolysis in vitro. Peripheral blood was obtained from 8 healthy subjects before and 1, 2, 4 and 8h after consuming a flavonoid-rich cocoa beverage that provided 0.25g/kg body weight (BW), 0.375 or 0.50g/kg BW of cocoa. Plasma flavanol and dimer concentrations were determined for each subject. Erythrocyte hemolysis was evaluated using a controlled peroxidation reaction. Epicatechin, catechin, 3'-O-methyl epicatechin and (-)-epicatechin-(4beta > 8)-epicatechin (Dimer B2) were detected in the plasma within 1 h after the consumption of the beverage. The susceptibility of erythrocytes to hemolysis was reduced significantly following the consumption of the beverages. The duration of the lag time, which reflects the capacity of cells to buffer free radicals, was increased. Consistent with the above, the purified flavonoids, epicatechin, catechin, Dimer B2 and the metabolite 3'-O-methyl epicatechin, exhibited dose-dependent protection against AAPH-induced erythrocyte hemolysis at concentrations ranging from 2.5 to 20 microM. Erythrocytes from subjects consuming flavonoid-rich cocoa show reduced susceptibility to free radical-induced hemolysis (p < 0.05).     

Polyethylene glycol additives reduce hemolysis in red blood cell suspensions exposed to mechanical stress

Mechanical damage to blood cells is of considerable concern in the development and use of circulatory assist devices and other blood contacting systems. Furthermore, hemodilution with saline, dextran, and other plasma expanders applied during extracorporeal circulation and dialysis increases red blood cell (RBC) susceptibility to the high shear stresses associated with these procedures. In this paper, we present polyethylene glycol (PEG) as a potential erythrocyte protective agent against mechanically induced cellular trauma. Bovine RBCs were subjected to mechanical stress induced by rolling stainless steel shots through RBC suspensions for a constant exposure time. The suspensions were prepared at a hematocrit of 30% in various media: PEG (20,000 molecular weight), autologous bovine plasma, Dextran 40 solution, and phosphate buffered saline (PBS). RBC suspensions in Dextran 40 were prepared at a viscosity similar to the PEG suspensions. We found the hemolysis level of RBCs suspended in plasma and in PEG solutions to be several times lower (p < 0.001) than in the Dextran and PBS solutions. No statistically significant difference was found between the hemolysis that occurred in suspensions of RBCs in autologous plasma and in 2.0% PEG solutions. Even PEG concentration as low as 0.1% reduced hemolysis by more than 40% compared with PBS or the same concentration of Dextran in suspension medium. Our data demonstrate the efficacy of PEG molecules in reducing mechanical trauma to erythrocytes and suggest the potential for using PEG in assisted circulation, dialysis, and other procedures where RBCs are subjected to extensive mechanical stress.     

APOPTOSIS IN HUMAN MONOCYTE-MACROPHAGES EXPOSED TO OXIDIZED LOW DENSITY LIPOPROTEIN

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca²⁺/Mg²⁺-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.