Reduced membrane fluidity and increased glycation of membrane proteins of platelets from diabetic subjects are not associated with increased platelet adherence to glycated collagen.

Platelets could contribute to vascular disease in diabetes through enhanced adherence to collagen exposed in injured vessels. Increased platelet adherence to collagen in diabetes could result from an alteration in platelets and/or platelet hypersensitivity to collagen that has been glycated to a greater extent. In this study, the adherence of platelets from diabetic or control subjects to glycated or nonglycated collagen coated onto glass surfaces was examined. Membrane fluidity of platelets was also determined, since decreased membrane fluidity associated with increased glycation of membrane proteins of platelets from diabetic subjects was shown in a previous study, and decreases in membrane fluidity have been shown by others to increase platelet adhesion. Thirteen diabetic subjects were compared with 13 age-and sex-matched control subjects. Collagen was glycated (9.7 nmol glucose/mg protein) by preincubation for 12 days in glucose-rich medium (500 mmol/L). A control solution of collagen incubated without glucose for the same time had 3.3 nmol glucose/mg protein. There were no differences in the adherence of platelets from diabetic and control subjects to nonglycated and glycated collagen-coated glass. The mean steady-state fluorescence polarization value (0.187 +/- 0.002) in 1.6-diphenyl-1,3,5-hexatriene-labeled platelets from diabetic subjects was significantly greater than in platelets from control subjects (0.174 +/- 0.002, p < 0.002); thus membrane fluidity in platelets from the group of diabetic subjects was decreased. The extent of glycation of membrane proteins from diabetic subjects (25.4 +/- 0.5 nmol glucose/mg protein) was significantly greater than from control subjects (20.2 +/- 0.4 nmol glucose/mg protein, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonenzymatic protein glycosylation. I. Lowered erythrocyte membrane fluidity in juvenile diabetes

In order to determine the fluidity of erythrocyte membrane ghosts the degree of random orientation of excited diphenylhexatriene molecules (anisotropy) was examined in normal and diabetic juveniles. A small but significant enhancement of diphenylhexatriene anisotropy was observed in diabetic erythrocyte membranes when compared with control (0.184 +/- 0.013 versus 0.152 +/- 0.010). This alteration was accompanied by an increase of nonenzymatic glycosylation of membrane proteins and haemoglobin in diabetic subjects (25.7 +/- 2.6 versus 21.1 +/- 2.2 and 7.1 +/- 1.4 versus 4.0 +/- 1.0 respectively). Moreover, elevated plasma glycaemia in diabetes was correlated with an increase in membrane anisotropy in the erythrocyte membranes of diabetic juveniles. Linear relationships were seen between glycosylated hemoglobin and glycosylated membrane proteins (r = 0.767) as well as between glycosylated haemoglobin and membrane anisotropy (r = 0.741). We suggest that membrane protein glycosylation enhancement occurring in diabetes could be one of the reasons of the lowered diabetic erythrocyte membrane fluidity.     

Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes.

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.     

Nonenzymatic glycation of human platelet membrane proteins in vitro and in vivo

Human platelet membrane proteins (PMP), incubated in vitro in the presence of various concentrations of glucose, undergo nonenzymatic glycation, as evidenced by incorporation of [3-3H]glucose radioactivity into the acid-precipitable fraction. The time course of the reaction is linear for the first hours, and the rate of glycation depends on the glucose concentration in the medium: at a glucose concentration of 80 mmol/L, up to 60 nmol of glucose is bound per milligram of PMP. The ketoaminic nature of the glucose/protein linkages was demonstrated by the finding of 5-hydroxymethylfurfuraldehyde by liquid-chromatographic analysis of acid hydrolysates of PMP. We analyzed PMP from 13 subjects with type I poorly controlled diabetes and from 10 nondiabetics. Nonenzymatic glycation, evaluated as nanomoles of the aldehyde per milligram of protein, was much greater in diabetic patients than in nondiabetics: 1.58 +/- 0.70 vs 0.37 +/- 0.18 (mean +/- SD).     

遗传性葡萄糖—6—磷酸脱氢酶缺乏症红细胞膜脂质的改变

目的：探讨葡萄糖－６－磷酸脱氢酶（Ｇ６ＰＤ）缺乏症溶血的生化机制。方法：采用荧光偏振法测定红细胞膜脂流动性；荧光分光光度法测定膜过氧化脂质（ＭＤＡ）；硅胶柱层析及薄板层析分离磷脂，气相色谱法分析磷脂中脂肪酸含量。结果：Ｇ６ＰＤ缺乏症荧光偏振度为０．２６３３±０．００４３，较正常对照的０．２２６１±０．００６９高（Ｐ＜０．０１）；Ｇ６ＰＤ缺乏时ＭＤＡ含量为１．４８２±０．０９５ｎｍｏｌ／ｍｇ膜蛋白，正常对照为０．３８２±０．０７２ｎｍｏｌ／ｍｇ膜蛋白（Ｐ＜０．０１）；Ｇ６ＰＤ缺乏症总磷脂及磷脂酰丝氨酸中不饱和脂肪酸Ｃ１８∶１，Ｃ１８∶２含量减少（Ｐ均＜０．０５）。结论：红细胞膜脂流动性降低可能是Ｇ６ＰＤ缺乏症溶血的一个重要原因。     

Effect of low-intensity (3.75-25 J/cm2) near-infrared (810 nm) laser radiation on red blood cell ATPase activities and membrane structure

OBJECTIVE: The biostimulation and therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the exact mechanism of action of the laser radiation with living cells is not yet understood. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75-25 J/cm(2)) on the structure of protein and lipid components of red blood cell membranes and it functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. BACKGROUND DATA: A variety of studies both in vivo and in vitro showed significant influence of laser irradiation on cell functional state. At the same time another group of works found no detectable effects of light exposure. Some different explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe biological effects of laser light. It was suggested that optimization of the structural-functional organization of the erythrocyte membrane as a result of laser irradiation may be the basis for improving the cardiac function in patients under a course of laser therapy. MATERIALS AND METHODS: Human red blood cells or isolated cell membranes were irradiated with low-intensity laser light (810 nm) at different radiant exposures (3.75-25 J/cm(2)) and light powers (fluence rate; 10-400 mW) at 37 degrees C. As the parameters characterizing the structural and functional changes of cell membranes the activities of Na(+)-, K(+)-, and Mg(2+)-ATPases, tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into membrane lipid bilayer were used. RESULTS: It was found that near-infrared low-intensity laser radiation changes the ATPase activities of the membrane ion pumps in the dose- and fluence rate-dependent manner. At the same time no changes of such integral parameters as cell stability, membrane lipid peroxidation level, intracellular reduced glutathione or oxyhaemoglobin level were observed. At laser power of 10 mW, an increase of the ATPase activity was observed with maximal effect at 12-15 J/cm(2) of light dose (18-26% for the total ATPase activity). At laser power of 400 mW (fluence rate significantly increased), inhibition of ATPases activities mainly due to the inhibition of Na(+)-, K(+)-ATPase was observed with maximal effect at the same light dose of 12-15 J/cm(2) (18-23% for the total ATPase activity). Fractionation of the light dose significantly changed the membrane response to laser radiation. Changes in tryptophan fluorescent parameters of erythrocyte membrane proteins and the increase in lipid bilayer fluidity measured by pyrene monomer/excimer fluorescence ratio were observed. CONCLUSIONS: Near-infrared laser light radiation (810 nm) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both erythrocyte membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membranes and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps and, thus, changes in membrane ion flows.     

The effect of simvastatin on erythrocyte membrane fluidity during oxidative stress induced by cardiopulmonary bypass: a randomized controlled study

BACKGROUND: Abnormal erythrocyte deformability can cause severe complications during cardiopulmonary bypass (CPB) surgery, including both hemolysis and perfusion abnormalities. OBJECTIVES: The goals of this study were to evaluate changes in erythrocyte membrane fluidity and lipid peroxidation during CPB and to examine the effect of simvastatin treatment on these parameters. METHODS: Patients undergoing cardiac surgery involving CPB were selected and randomized to receive either simvastatin 40 mg/d or placebo for 3 weeks before surgery. Three blood samples were obtained at different times during surgery for analysis of erythrocyte membrane fluidity, anion permeability, and lipid peroxidation. Erythrocyte ghosts were prepared and incubated with a lipophilic fluorescent probe (diphenyl-hexatriene), and fluorescence anisotropy was evaluated by spectrophotofluorimetric assay as a measure of membrane fluidity. Anion permeability was evaluated by the specific absorption of methemoglobin (CM) at 590 and 635 nm after treatment of heparinized blood with NaNO2. The formation of thiobarbituric acid-reactive substances was evaluated as an index of lipid peroxidation. Aspartate transaminase and lactate dehydrogenase were also measured as indices of hemolysis. RESULTS: Forty patients met the inclusion criteria (20 simvastatin, 20 placebo). Their characteristics differed significantly at baseline only in terms of the lipid profile; the statin group had higher levels of high-density lipoprotein cholesterol (P = 0.01) and lower levels of low-density lipoprotein cholesterol (P = 0.001) than the placebo group. CPB was found to significantly modify characteristics of the erythrocyte membrane. Compared with preoperative values, CPB induced decreases in both mean (SD) erythrocyte membrane fluidity and anion permeability (preoperative CM: 0.69 [0.02]; 24-hour postoperative CM: 0.18 [0.02]; P < 0.001) and an increase in mean (SD) membrane lipid peroxidation (preoperative malonyl dialdehyde [MDA]: 0.21 [0.01] nmol/mL; postoperative MDA: 0.10 [0.02] nmol/mL; P < 0.001). Treatment with simvastatin was associated with a significant reduction in mean (SD) membrane lipid peroxidation both preoperatively and at 24 hours postoperatively compared with placebo (preoperative MDA: 0.07 [0.01] vs 0.10 [0.02] nmol/mL, respectively; P < 0.05; postoperative MDA: 0.10 [0.04] vs 0.21 [0.01] nmol/mL; P < 0.05). In addition, statin treatment was associated with significant increases in anion permeability preoperatively and postoperatively compared with placebo (preoperative CM: 0.79 [0.01] vs 0.69 [0.02]; P < 0.01; 24-hour postoperative CM: 0.30 [0.01] vs 0.18 [0.02]; P < 0.01). CONCLUSION: The results of this study suggest that among these patients undergoing CPB surgery, use of simvastatin for 3 weeks before the surgery had significant beneficial effects on erythrocyte membrane fluidity, lipid peroxidation, and anion permeability.     

Modifications induced by diabetes on the physicochemical and functional properties of erythrocyte plasma membrane

Increasing evidence suggests that in experimental diabetes an impairment in Na+,K+-ATPase activity plays a central role in the pathophysiology of diabetic complications, while only a few data are available with regard to human subjects. We studied the erythrocyte membrane Na+,K+-ATPase activity and membrane fluidity in insulin-dependent and non-insulin-dependent diabetic subjects. A significant decrease in the enzyme activity and in fluorescence polarization values was found in both groups compared with normal subjects. Neither Na+,K+-ATPase activity nor membrane fluidity was found to be related to metabolic control, assessed by means of fasting blood glucose levels and HbA1c. On the contrary, a significant correlation was observed between Na+,K+-ATPase activity and membrane fluidity in both insulin-dependent and non-insulin-dependent diabetic subjects. The present work provides evidence that a reduction in the Na+,K+-ATPase activity is present in the plasma membranes of insulin-dependent and non-insulin-dependent diabetics. Furthermore, it suggests that the change in enzyme activity might be related to modifications in membrane fluidity.     

D-lysine effectively decreases the non-enzymic glycation of proteins in vitro

Excessive non-enzymic glycation of proteins alters their physicochemical properties, with possible pathological effects. We investigated the in vitro inhibition of protein glycation by D-lysine--an isomer not incorporated into mammalian proteins but possessing the same chemical characteristics as L-lysine. Glucose incorporation was studied as follows: (a) human albumin, IgG, collagen, and isolated glomerular basement membrane were incubated for 20 days with D-glucose (5.0, 10.0, and 20.0 mmol/L) in the presence of D-lysine at 1/10 the sugar concentration; (b) albumin was incubated in similar glucose concentrations but with a constant amount (2.0 mmol/L) of D-lysine; (c) albumin and IgG were incubated for 10 days in buffer containing glucose (10 mmol/L) and increasing concentrations of D-lysine (0.25, 0.5, 1.0, 2.0, and 4.0 mmol/L); (d) inhibition specificity was tested by treating albumin as in c but with glycerol present rather than D-lysine. In addition, we measured ketoamine after incubating albumin (50 g/L) in 10 mmol/L glucose for 10 days in the presence of D-lysine (0.25, 0.5, 1.0, and 2.0 mmol/L). The results show that (a) the amount of glucose bound to the four proteins was significantly (P less than 0.05) decreased in the presence of D-lysine at the higher concentrations of glucose; (b) the lower the glucose concentration, the higher was the inhibitory effect of D-lysine; (c) the inhibition of glucose incorporation into proteins correlated directly with the concentration of D-lysine; (d) no inhibition was observed with glycerol. Ketoamine decreased with increase in D-lysine (P less than 0.01). The effective diminution of non-enzymatic glycation by D-lysine highlights its potential use in vivo.     

Gallium-inducible transferrin-independent iron acquisition is a property of many cell types: possible role of alterations in the plasma membrane.

BACKGROUND: We have previously shown that human myeloid cell types can acquire large amounts of iron (Fe)3+ from low-molecular-weight chelates by a process that is independent of adenosine triphosphate and dramatically increased by gallium (Ga) and other multivalent cationic metals. METHODS: To provide further insight into the mechanism responsible and its relevance to other cellular systems, we investigated Fe acquisition from nitrilotriacetic acid (NTA) by several myeloid and nonmyeloid cell lines in the presence and absence of Ga. RESULTS: Most nonmyeloid cells examined exhibited similar ability to acquire Fe from NTA. Ga increased the apparent maximum velocity (Vmax), with minimal changes in apparent Michaelis constant (Km), of all cell lines. Both erythrocytes and erythrocyte ghosts acquired Fe from NTA, which increased with Ga exposure, analogous to nucleated cells. However, liposomes made from phospholipids did not exhibit Ga-inducible Fe association. Enzymes that modify surface proteins and carbohydrates did not alter HL-60 cell Fe acquisition. Modifying HL-60 membrane fatty acid content had only a minimal effect. Ga exposure did not change membrane potential or fluidity. However, electron microscopy suggested that Ga alters plasma membrane physical properties. CONCLUSION: Multivalent cations appear to induce changes in cell membranes that may alter their interaction with Fe3+ and probably other multivalent cations.     

Ethnic differences in erythrocyte membrane fluidity and the association with serum triacylglycerols

The objectives of this study were to determine whether there are differences between black and white individuals with regard to the membrane fluidity of isolated erythrocytes, and/or in the relationships between membrane fluidity, gender and circulating lipids. Fluorescent polarization anisotropy, as an index of membrane fluidity, was determined using the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in 52 black and 52 white individuals, of whom 39 pairs were matched for age, sex and blood pressure. In the 39 matched pairs, the TMA-DPH anisotropy was significantly higher in the black (0.262+/-0.007) compared with the white (0.258+/-0.005) subjects (P<0.005). There was also a significant difference in serum lipids. Gender differences in TMA-DPH anisotropy were observed in the white but not in the black individuals. The associations between membrane fluidity and serum lipids were examined in the total group, separated according to ethnic group. Although the associations were in the same direction in both groups, the association was only significant in the white subjects (r= - 0.42; P<0.02). The ethnic difference in membrane fluidity was abolished when adjusting for serum triacylglycerols. In conclusion, ethnic differences in erythrocyte membrane fluidity, as determined by the use of TMA-DPH anisotropy, appear to be the result of ethnic differences in the level of serum triacylglycerols.     

Erythrocyte membrane fluidity in adult polycystic kidney disease: difference between intact cells and ghost membranes

In adult polycystic kidney disease (APCKD) the cytoskeleton of renal tubular cells is abnormal. In erythrocytes the cytoskeleton affects the fluidity of membrane lipids. The authors determined fluorescence anisotropy in intact erythrocytes and erythrocyte ghosts in 12 APCKD patients and 12 normal subjects. In APCKD whole erythrocytes had a much lower core-region anisotropy, which indicated higher membrane fluidity than normal (mean 0.175 vs. 0.224, P  < 0.01). This abnormality was not detected in erythrocyte ghosts, which suggests that preparation of ghosts altered membrane lipid organization. This could be directly due to ghosting or secondary to the loss of cytoskeletal effects, which may be abnormal in APCKD.     

Physicochemical and functional modifications induced by obesity on human erythrocyte membranes

BACKGROUND: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. DESIGN: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. RESULTS: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. CONCLUSION: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.     

Role of liver plasma membrane fluidity in the pathogenesis of estrogen-induced cholestasis

The role of liver plasma membrane (LPM) fluidity in the pathogenesis of intrahepatic cholestasis in rats was assessed by comparing the effects of ethinyl estradiol, a cholestatic agent, and spironolactone on membrane fluidity and bile flow. Spironolactone is a steroid that has some feminizing actions but that lacks the phenolic A ring necessary for estrogens to cause cholestasis. Bile flow was reduced 42% (p less than 0.01) by ethinyl estradiol and increased 22% (p less than 0.05) by spironolactone; however, both agents produced a significant reduction of membrane Na+, K+-ATPase activity (p less than 0.01) and fluidity (p less than 0.01). The decreased fluidity persisted in liposomes prepared from the total lipid extract as well as the phospholipid extract of these membranes. Both agents produced similar significant increases in the cholesterol ester content and cholesterol-to-phospholipid molar ratio of the membranes. In addition, ethinyl estradiol and spironolactone increased the membrane sphingomyelin content (15% and 11%, respectively); however, neither agent altered the fatty acid composition of the phospholipids. Because the decreased fluidity persisted in liposomes prepared from phospholipids extracted from the LPMs of treated rats, changes in membrane cholesterol are not the sole cause of the altered membrane fluidity. Rather, the increased sphingomyelin is at least partially responsible for these changes. Also, because ethinyl estradiol and spironolactone produce similar changes in LPM lipid composition and fluidity but disparate effects on bile flow, membrane fluidity as assessed by fluorescence polarization does not appear to be the rate-limiting determinant of bile flow in estrogen-induced cholestasis.     

Abnormal degradation of erythrocyte membrane proteins in hereditary spherocytosis

The degradation rate of erythrocyte membrane proteins by membrane-bound proteases was compared in healthy controls and in patients with hereditary spherocytosis (HS). An increased degradation rate of spectrin and a decreased digestion rate of Band 3 were found in HS patients. These differences may be due to altered accessibilities of membrane proteins to proteases and/or to changes in the pattern of membrane-associated proteases and may be connected with the decrease in the spectrin: Band 3 ratio reported for erythrocyte membranes in HS.     

Platelet membrane fluidity and peroxynitrite levels in migraine patients during headache-free periods

Previous investigations have suggested that changes in platelet activity may play a key role in the pathophysiology of migraine via mechanisms involving the nitric oxide (NO) pathway. Changes in platelet response and nitrite levels have recently been demonstrated during migraine attacks, while there is considerable uncertainty about NO activity in headache-free periods. A reactive oxidant produced from NO and superoxide anion at the site of inflammation, peroxynitrite (ONOO-) has effects including changes in membrane activity and fluidity. The aim of the present study was to determine ONOO- levels in the platelets of patients suffering from migraine during the headache-free period. Nitric oxide synthase (eNOS and iNOS) expression in platelets and the effects of ONOO- on membrane Na+/K+-ATPase activity and membrane fluidity were also evaluated. Subjects were 57 patients suffering from migraine without aura and 35 controls. Blood samples were collected in the headache-free period. Platelet ONOO- levels were determined using dichlorofluorescein acetate with steady-state fluorescence. Platelets were then probed for induction of eNOS and iNOS expression by western immunoblotting. Membrane Na+/K+-ATPase activity and fluidity were determined with the fluorescent probes TMA-DPH and DPH. In the presence of extracellular l-arginine(100 micromol/l), ONOO- production was significantly greater in patients' platelets than in those of controls (P < 0.001). Western immunoblotting of platelet proteins evidenced higher iNOS expression in patients than in controls. In addition, platelet membrane Na+/K+-ATPase activity and membrane fluidity evaluated by TMA-DPH were significantly lower in patients (P < 0.001). In conclusion, migraine patients show intercritic changes in platelet membrane fluidity and activity that may be related to the oxidative stress caused by increased ONOO- levels.     

Effect of lipid peroxides and antioxidants on glycation of hemoglobin: an in vitro study on human erythrocytes

BACKGROUND: Glycation and lipid peroxidation are two important processes known to play a key role in complications of many pathophysiological process. We sought to assess the possibility of an interaction between these processes in vitro and to examine the effect of lipoic acid and taurine on the glycation of hemoglobin and lipid peroxidation. METHODS: Human erythrocytes in phosphate buffered saline (pH 7.4) were incubated with 5 or 50 mmol/l glucose. To study the effect of antioxidants on glycation of hemoglobin, erythrocytes were incubated with either lipoic acid or taurine and then exposed to glucose concentration of either 5 or 50 mmol/l. To clarify if lipid peroxides per se enhances the glycated hemoglobin level, an in vitro study was performed by incubating erythrocyte suspension containing either 5 or 50 mmol/l glucose with or without MDA. Lipid peroxides and glycated hemoglobin levels were determined in the glucose treated cells. RESULTS: Glycated hemoglobin levels were higher in erythrocytes incubated with 50 mmol/l glucose concentrations than in erythrocytes incubated with 5 mmol/l glucose. The increase in glycated hemoglobin levels was blocked significantly when erythrocytes were pretreated with either lipoic acid or taurine. Both the antioxidants used in the present study markedly reduced the MDA levels. The level of glycated hemoglobin in erythrocyte incubated in the presence of MDA was increased significantly when compared to erythrocyte incubated with glucose alone. CONCLUSIONS: Lipid peroxides per se may have a role to play in glycation of hemoglobin and antioxidants (lipoic acid and taurine) can partially inhibit the formation of glycated hemoglobin by lowering the levels of lipid peroxides.     

Biochemical assessments of oxidative stress, erythrocyte membrane fluidity and antioxidant status in professional soccer players and sedentary controls

BACKGROUND: Physical exercise is characterized by an increase in oxygen consumption by the whole body. This leads to a decrease in antioxidant levels that could promote both an increase in the markers of lipoprotein peroxidation and damage to the erythrocyte membrane with consequent modification of membrane fluidity. MATERIALS AND METHODS: Different markers of oxidative stress, erythrocyte membrane fluidity and antioxidant status were determined in 20 professional soccer players and 20 sedentary controls. Plasma lipoperoxides and kinetics of Cu-stimulated plasma peroxidation were measured together with hydrosoluble (albumin, uric acid and vitamin C), liposoluble (vitamin E and bilirubin) and enzymatic (superoxide dismutase and glutathione peroxidase) serum antioxidants. Erythrocyte membrane rigidity was determined by measuring fluorescence anisotropy (rs) of the fluorescent probe 1, 3, 5 diphenylexatriene. RESULTS: The sportsmen showed higher levels of the following plasmatic antioxidants: ascorbic acid (P<0.0001), uric acid (P<0.0001), alpha-tocopherol (P=0.03) and superoxide dismutase activity (P=0.0001). According to this evidence, the lipoperoxide levels (P=0.0158), the duration of the latency phase of plasma peroxidation (P=0.0123) and erythrocytes membrane fluidity (P=0.0152) were found to be significantly higher in the soccer players. DISCUSSION: Athletes undergoing regular and adequate training show improved antioxidant status together with a more fluid membrane status, which could contribute to improving both peripheral resistance to insulin and all the functional metabolic interchanges in the cellular membrane.     

Drug-induced erythrocyte membrane internalization

In vitro erythrocyte membrane internalization, resulting in the formation of membrane-lined vacuoles, can be quantified by a radioisotopic method. A complex of (37)Co-labeled vitamin B(12) and its plasma protein binders is first adsorbed to the cell surface, and after vacuoles are formed, the noninternalized label is removed by washing and trypsin treatment. The residual radioactivity represents trapped label and can be used to measure the extent of membrane internalization.Using this method, it was found that in addition to primaquine, a group of membrane-active drugs, specifically hydrocortisone, vinblastine, and chlorpromazine can induce membrane internalization in erythrocytes. This is a metabolic process dependent on drug concentration, temperature, and pH. Vacuole formation by all agents tested can be blocked by prior depletion of endogenous substrates or by poisoning the erythrocytes with sodium fluoride and sulfhydryl blocking agents. This phenomenon resembles in some respects the previously reported membrane internalization of energized erythrocyte ghosts. It is suggested that membrane internalization is dependent on an ATP-energized state and is influenced by the balance between the concentrations of magnesium and calcium in the membrane. This study provides a basis for proposing a unifying concept of the action of some membrane-active drugs, and for considering the role of erythrocyte membrane internalization in pathophysiologic events.     

Peripheral lymphocyte membrane fluidity after thermal injury.

Serum cortisol levels are increased in patients after thermal injury. Lymphocyte function is altered in these patients, which renders them susceptible to infections. Elevated cortisol levels may contribute to this compromised state. In this study, we have demonstrated that cortisol directly affects lymphocyte membrane fluidity as measured by the polarization of fluorescence from the membrane-associated probe diphenylhexatriene in peripheral blood lymphocytes. Membrane fluidity increased in vitro with short- or long-term cortisol exposure. However, membranes of control peripheral blood lymphocytes that were previously exposed to cortisol became resistant to the fluidizing effect of cortisol, which implies membrane adaptation to long-term cortisol exposure. Cortisol effects were similar to those associated with ethanol, a known membrane-fluidizing agent, in peripheral blood lymphocytes and cytotoxic T lymphocytes. Membrane fluidity was compared in peripheral blood lymphocytes from thermally injured patients and peripheral blood lymphocytes from normal (control) subjects. Peripheral blood lymphocyte membrane fluidity increased in major thermal injury. Our data suggest that cortisol affects lymphocyte membrane fluidity in vitro in a manner similar to the membrane fluidity alterations that are observed in vivo after thermal injury. These observations reflect a direct membrane effect of cortisol, which may explain, in part, the cellular dysfunction and immunologic suppression that is observed after thermal injury.