Fatty acids composition in erythrocyte membranes of athletes after one and after a series of whole body cryostimulation sessions

Whole body cryotherapy (WBC) is a treatment often used by athletes as part of biological renewal. Despite the large interest in this form therapy there is still a lack of information on the effects of WBC on the concentration of fatty acids in erythrocyte membranes. Our study aimed at comparing the fatty acids (FA) composition of erythrocyte membranes of athletes after one session and after a series of sessions of whole body cryostimulation. In our study small changes in the level of total cholesterol (decrease) were observed 24 h after a single session. After the twelfth session of whole body cryostimulation, the level of saturated fatty acids (SFA), mainly palmitic acid (C16:0) and n-3 fatty acid eicosapentaenoic (EPA, C20:5n-3) increased almost two-times fold in the red blood cell membranes. The level of n-6 polyunsaturated fatty acids (PUFA n-6), mainly gamma-linolenic acid (C18:3n-6) as well as trans fatty acids (elaidic acid) decreased in the erythrocyte membranes from men after a series of session in a cryochamber, when compared to the control sample. The n-3/n-6 FA ratio in the erythrocyte membranes was higher after twelfth session in a cryochamber in comparison to the control sample.The data obtained during our study will be important for further research regarding the biochemistry of lipids in men after sessions of whole body cryostimulation.     

Membrane fluidity of platelets and erythrocytes in patients with Alzheimer's disease and the effect of small amounts of aluminium on platelet and erythrocyte membranes

The membrane fluidity of platelet and erythrocyte membranes in 10 Alzheimer's disease patients and 9 age-matched controls was studied. The platelet membranes of patients with Alzheimer's disease were found to be significantly more fluid than those of controls (p<0.02). However, erythrocyte membranes of Alzheimer patients were less fluid (more viscous) than those of controls (p<0.05). On further investigation of platelet and erythrocyte membranes obtained from healthy volunteers, the fluidity was found to change with increasing aluminium concentrations. When aluminium ammonium sulphate (0.01–10 M) was added to membrane suspensions, the fluidity of platelet membranes was increased, whereas the fluidity of erythrocyte membranes was decreased (i.e. the microviscosity was increased).     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl₃ was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 μM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 μM to 100 μM.     

Effects of dietary lead,cadmium, mercury,and selenium on fatty acid composition of blood serum and erythrocyte membranes from chicks

A series of experiments was conducted to study the effects of dietary and injected lead (as Pb acetate-3H₂O) and of dietary Cd, Hg, and Se on fatty acid composition of serum lipids of chicks as measured by gas-liquid chromatography. The effect of dietary Pb on fatty acid composition of erythrocyte membranes was measured also. Dietary Pb (1000 ppm) increased the serum concentration of arachidonic acid (20:4, first no. = no. of carbon atoms:second no. = no. of double bonds) and decreased the concentration of linoleic acid (18:2) and the ratio 18:2/20:4. Intraperitoneal injection of Pb (52 mg/100 g body weight) did not alter serum fatty acid composition by 4 h post-injection. The separate effects of 2000 ppm Pb, 60 ppm Cd, 500 ppm Hg, and 10 ppm Se added to the diet on serum fatty acids were measured in a single experiment. In comparison to controls, Pb and Cd lowered serum concentration of 18:2. Only Pb raised serum 20:4. Pb lowered the ratio 18:2/20:4, whereas Cd and Hg raised the ratio and Se was without effect. Dietary Pb (2000 ppm) raised the concentration of 20:4 and lowered the ratio 18:2/20:4 in erythrocyte membranes. The different effects of injected and dietary Pb on the serum 18:2/20:4 ratio suggest that Pb alters 20:4 synthesis from 18:2 rather than mobilization of 20:4 from tissues. The Pb-induced increase of lipid peroxida-tion in erythrocytes observed by other workers may be a reflection of increased 20:4 level in erythrocyte membranes.     

Effect of a hyperlipidic diet on lipid composition, fluidity, and (Na+-K+)ATPase activity of rat erythrocyte membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.     

Interaction of corundum and quartz nanocrystals with erythrocyte membranes

Mechanisms of interaction of quartz and corundum nanocrystals with erythrocyte membranes were studied by means of atomic force microscopy and fluorescence analysis. Hydrophobic, chemically inert nanocrystals larger than a critical size (20–25 nm) can bind to erythrocyte membranes without damaging them. If the size of the nanocrystals is less than 15 nm, they can penetrate into the lipid bilayer membranes. This decreases the membrane microviscosity, and pores appear, which leads to cell lysis. A thermodynamic explication of the critical size of the nanocrystals is given.     

Changes in the fatty acid composition of blood cell membranes in children with inflammatory diseases

Fatty acid composition of erythrocyte and leukocyte membranes has been studied in children (with normal body mass and obesity) with inflammatory diseases of the gastrointestinal tract and children with bronchial asthma in comparison with basically healthy children. Fifty seven children aged from 7 to 14 years were examined: 13 with inflammatory diseases of the gastrointestinal tract (IDGIT) (eosophagitis, gastroduodenitis, stomach ulcer), 25 with obesity stages (I–III) complicated by IDGIT, 9 with bronchial asthma and 10 basically healthy children. The study revealed that both IDGIT and bronchial asthma caused significant and similar changes in fatty acid composition of cell membranes. These included accumulation of ω3 eicosapentaenoic acid (EPA) and the decrease of docosahexaenoic acid (DHA); this phenomenon observed in both erythrocyte and leukocyte membranes suggests a common feature of the detected changes in fatty acid composition of cell membranes in inflammation. There was a significant decrease in the level of membrane ω6 polyunsaturated fatty acids (PUFA), first of all arachidonic acid and total ω6 PUFA. Consequently, EPA accumulation in membranes may be a compensatory response to low dietary arachidonic acid supply and/or its increased loss for the synthesis of pro-inflammatory eicosanoids (prostaglandins, leukotriens, thromboxans) during inflammatory process.     

Thermotropic lipid phase separations in human erythrocyte ghosts and cholesterol-enriched rat liver plasma membranes

Summary Electron spin resonance (ESR) studies of human erythrocyte ghosts labeled with 5-nitroxide stearate, I(12,3), indicate that a temperature-dependent lipid phase separation occurs with a high onset at 38°C. Cooling below 38°C induces I(12,3) clustering. Similar phase separations were previously identified in human platelet and cholesterol-loaded [cholesterol/phospholipid molar ratio (C/P)=0.85] rat liver plasma membranes [L.M. Gordon et al., 1983;J. Membrane Biol. 76; 139–149]; these were attributed to redistribution of endogenous lipid components such that I(12,3) is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains. Further enrichment of rat liver plasma membranes to C/P ratios of 0.94–0.98 creates an artificial system equivalent to human erythrocyte ghosts (C/P=0.90), using such criteria as probe flexibility, temperature dependent I(12,3) clustering; and polarity of the probe environment. Consequently, cholesterol-rich and-poor domains probably exist in both erythrocyte ghosts and high cholesterol liver membranes at physiologic temperatures. The temperature dependence of cold-induced hypertonic lysis of intact human erythrocytes was examined by incubating cells in 0.9m sucrose for 10 min at 1°C intervals between 9 and 46°C (Stage 1), and then subjecting them to 0°C for 10 min (Stage 2). Plots of released hemoglobin are approx. sigmoidal, with no lysis below 18°C and maximal lysis above 40°C. The protective effect of low temperatures during Stage 1 may be due to the formation of cholesterol-rich domains that alter the bilayer distribution and/or conformation of critical membrane-associated proteins.     

Damage to the structure of erythrocyte plasma membranes in patients with type-2 hypercholesterolemia

BACKGROUND: Hypercholesterolemia may decrease the deformability of red blood cells which impairs their hemorheological behavior and promotes atherosclerosis.The study involved 60 hypercholesterolemic patients and 30 healthy individuals as the control group.METHODS: The membrane fluidity of erythrocytes was estimated by a spin-label method (5-doxylstearic acid (5-DSA)). The ratio of weakly to strongly (W/S) immobilized residues of erythrocyte membrane-bond maleimide-tempo spin label was studied in oxidative damage to membrane protein. Damage to erythrocyte proteins was also indicated by means of Na(+) K(+) ATPase activity.RESULTS: The membranes of hyperlipidemia (hlp) patients contain larger concentrations of cholesterol 2.16+/-0.24 than do those of the normolipemic individuals 0.31+/-0.24 (P<0.001). The level of Na(+) K(+) ATPase in the erythrocyte membrane from the control group was higher 103.4+/-1.3 (nmolPi/(mgproteinsh)) than in the patient group 93.6+/-3.2 (nmolPi/(mgproteinsh)) (P<0.001). The order parameter S 5-DSA in the control group was 0.745+/-0.009 and in hlp patients was 0.755+/-0.009 (P<0.001). The W/S ratio in the control group amounted to 2.00+/-0.09 and in the hlp patient group was 2.50+/-0.11 (P<0.001).CONCLUSION: Type-2 hypercholesterolemia causes changes in the structure and fluidity of erythrocyte plasma membranes since the excess of cholesterol affects the normal rheology of blood through its interaction with erythrocytes. It also impairs the function and structure of plasma membrane proteins.     

The mobility and reactivity of maleimide-binding proteins in the rat erythrocyte membrane. Effects of dietary zinc deficiency and incubation with zinc in vitro

Erythrocyte ghosts, prepared from rats fed zinc-deficient diets, were analyzed for the mobility of membrane proteins by electron spin resonance spectroscopy of the sulfhydryl-binding spin probe, 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Compared with erythrocyte membranes from rats fed zinc-adequate diets ad libitum or pair-fed, erythrocyte membranes from zinc-deficient rats had a significantly increased ratio of weakly immobilized to strongly immobilized probe-binding proteins. This suggests that dietary zinc deficiency causes a conformational change in erythrocyte membrane proteins. Dietary zinc deficiency did not significantly affect N-ethylmaleimide (NEM)-induced thermal sensitivity or NEM-induced mechanical fragility in rat erythrocytes; however, the addition of zinc in vitro to red cells significantly inhibits NEM-induced mechanical fragility.     

Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes

The increased generation of reactive oxygen species that occurs in the condition of obesity may be responsible for oxidative injury to erythrocyte membranes, which could lead to a decrease in tissue oxygenation. Therefore, we have looked into the effects of obesity on both indexes of oxidative damage and physical-chemical properties of erythrocyte membranes in 50 overweight or obese [25 < body mass index (BMI) < 33], normotensive, nondiabetic women and 50 age-matched lean healthy women (BMI < 25). In the obese group compared with the lean group, we found that a) the onset of free radical-induced erythrocyte hemolysis and the ratio between reduced and oxidized glutathione were reduced, whereas the rate of free radical-induced damage increased; b) the n-3 fatty acid and the phospholipid contents decreased; c) the ratio between cholesterol and phospholipids increased; and d) the membrane fluidity decreased. These findings suggest an impairment of erythrocyte membrane physical-chemical properties in overweight and obese people as a consequence of oxidative injury that might be part of a pathogenetic mechanism responsible for obesity-related pathologies such as atherosclerosis and hypertension.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

Structural and functional changes in erythrocyte membranes in experimental atherosclerosis

Structural and functional characteristics of erythrocyte membranes were studied in rabbits with experimental atherosclerosis. In animals with single lipid spots in the aorta, a significant rise of the plasma cholesterol level was associated with the increased cholesterol/phospholipid (CS/PL) ratio and diminished activity of erythrocyte membrane Na+, K+-ATPase. EPMR spin probe data point to changes in structural membrane characteristics--an increase in order parameter for fatty acid chains of lipids and expansion of the temperature interval of the transition phase in the membranes. In rabbits with total aorta injury, a further increase both in the plasma cholesterol concentration and in the CS/PL ratio as well as in structural changes in erythrocyte membranes does not lead to another decrease in the enzymatic activity. In aorta homogenates of the experimental animals, the activity of Na+, K+-ATPase correlated with that in the erythrocyte membrane. This suggests the existence of similar chemical and structural changes in aorta cell membranes. The data may provide an indirect evidence in favour of the hypothesis of the involvement of smooth muscle cells and membrane enzymatic activity alterations in atherosclerosis.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

Changes in the structural characteristics and ATPase activity of the erythrocyte membranes in angiographically verified coronary artery lesion

Structural characteristics and Na, K-ATPase activity of erythrocyte membranes were studied by the spin probe method in patients with an angiographically verified damage to the coronary arteries. The rise of the cholesterol/phospholipid ratio in patients with coronary heart disease was associated with the increased orderliness of the fatty acid chains of erythrocyte membrane lipids, leading to inhibition of Na, K-ATPase activity. The data point to the same line of changes in erythrocyte membranes and smooth muscle cells during the development of coronary heart disease.     

Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects

Oxidative stress may play a role in the pathogenic mechanism of essential hypertension. Lipid peroxidation can alter the cellular structure of membrane-bound enzymes by changing the membrane phospholipids fatty acids composition. We investigated the relationship between (Na + K)-ATPase activity, lipid peroxidation, and erythrocyte fatty acid composition in essential hypertension. The study included 40 essential hypertensive and 49 healthy normotensive men (ages 35–60 years). Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking, and any current medication. Patients underwent 24-h ambulatory blood pressure monitoring and blood sampling. Lipid peroxidation was measured in the plasma and erythrocytes as 8-isoprostane or malondialdehyde (MDA), respectively. Antioxidant capacity was measured as ferric reducing ability of plasma (FRAP) in the plasma and as reduced/oxidized glutathione (GSH/GSSG ratio) in erythrocytes. (Na + K)-ATPase activity and fatty acids were determined in erythrocyte membranes. Hypertensives had higher levels of plasma 8-isoprostane, erythrocyte MDA, and relative percentage of saturated membrane fatty acids, but lower plasma FRAP levels, erythrocyte GSH/GSSG ratio, (Na + K)-ATPase activity and relative percentage of unsaturated membrane fatty acids, compared with normotensives. Day-time systolic and diastolic blood pressures correlated positively with lipid peroxidation parameters, but negatively with (Na + K)-ATPase activity. These findings suggest that the modulation of (Na + K)-ATPase activity may be associated with changes in the fatty acid composition induced by oxidative stress and provide evidence of a role for this enzyme in the pathophysiology of essential hypertension.     

Viscosity changes of erythrochyte membrane and membrane lipids at transition temperature

A non-linearity in the changes of viscosity with temperature was found in sonicated human erythrocyte membranes at 18–19 °C. At the same temperature, a break was observed in the viscosity of the extracted membrane lipids, the cholesterol content of which was varied by means of Sephadex LH 20 column chromatography. It is inferred that the break observed in the membranes corresponds to the transition temperature of the erythrocyte membrane lipids. The applied method of direct viscosimetry is relatively simple and cheap in comparison to the well known methods of ESR spectroscopy or differential scanning calorimetry, which have been hitherto widely used in determining thermal transition points in different systems.Viscosity measurements may be compared to light scattering or fluorescence measurements, introduced recently for the determination of phase transitions (Träuble, H. (1971) Naturwissenschaften 58, 277–284, and Lussan, C. and Faucon, J.F. (1971) FEBS Lett. 19, 186–188).     

Membrane lipid diffusion and band 3protein changes in human erythrocytes due to acute hypobaric hypoxia

Because it has been reported that hypoxia in rats may promotelipid peroxidation and other free radical reactions that could modifymembrane lipids and proteins, the effect of acute hypobaric hypoxia onhuman erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusionstatus in the membrane bilayer. Membrane protein modification wasdetected by SDS-PAGE. Healthy young men were exposed for 20 min to thehypobaric hypoxia, simulating an altitude of 4,500 m. Under thiscondition, erythrocyte membrane lipids reached a state of higherlateral diffusivity with respect to normobaric conditions and membraneband 3 protein was modified, becoming more susceptible tomembrane-bound proteinases. These observations suggest that acutehypobaric hypoxia may promote an oxidative stress condition inthe erythrocyte membrane.