Effects of long-chain acyl carnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitines accumulate in ischemic myocardium and potentially contribute to the myocardial damage. To characterize alterations in membrane molecular dynamics produced by palmitoylcarnitine, human erythrocytes were spin-labeled with 5-doxylstearic acid, and membrane fluidity was quantified by measuring the changes in the order parameter derived from ESR spectra. Palmitoylcarnitine induced triphasic alterations in membrane fluidity of human erythrocytes. The membrane fluidity increased for 5 min, then decreased in a concentration-dependent manner. At higher concentrations (100 and 150 microM) of palmitoylcarnitine, membrane fluidity increased again after 30 and 20 min of the incubation, respectively. Addition of 2.8 mM CaCl2 resulted in a significant decrease in membrane fluidity and enhanced the alterations in membrane fluidity caused by palmitoylcarnitine. The results suggest that alterations in molecular dynamics are one mechanism through which long-chain acyl carnitine could play an important role in ischemic injury.     

Effects of L-carnitine and palmitoylcarnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitine accumulate in ischemic myocardium and potentially contribute to the myocardial damage, and the role of carnitine in protecting the heart against ischemic damage is interesting. It has been reported that palmitoylcarnitine causes alterations in the membrane molecular dynamics, so this study was designed to investigate whether L-carnitine had a stabilizing effect of membrane fluidity using the spin-label technique. Human erythrocytes were spin-labeled with 5-doxylstearic acids, and membrane fluidity was quantified by measuring the change in the order parameter S. The administration of palmitoylcarnitine (100 microM) altered the membrane fluidity of erythrocytes and caused significant morphological changes. L-carnitine (2mM) decreased the alteration of the fluidity of erythrocytes incubated with palmitoylcarnitine (100 microM), and improved the morphological changes in erythrocytes. These results show that L-carnitine has a stabilizing effect of membrane fluidity as a result of interaction with the palmitoylcarnitine which has a detergent effect.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

The synthesis of palmitoylcarnitine by etio-chloroplasts of greening barley leaves

CoASH, Mg²⁺, ATP and (-)-carnitine were found to be essential for the production of palmitoylcarnitine from palmitate by purified barley etio-chloroplasts. It was concluded that long-chain acyl CoA synthetase (palmitoyl CoA synthetase, EC 6.2.1.3) and carnitine long-chain acyl-transferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity were present in the etio-chloroplasts. It is suggested that the long-chain acylcarnitine formed may move more easily through membrane barriers than the long-chain acyl CoA compound. Also or alternatively this enzyme may spare CoA by transferring long-chain acyl groups from long-chain acyl CoA to carnitine.     

脂质过氧化对人红细胞膜脂流动性的影响

研究枯稀过氧化氢/高铁血红素体系所产生的烷基过氧自由基对红细胞的损伤。测定了脂质过氧化的产物——丙二脂的生成,并证明阿魏酸钠对脂质过氧化的抑制。荧光偏振的结果指出,膜脂过氧化以后降低了膜脂的流动性。人红细胞用5DSA和16DSA标记并用ESR检测膜脂流动性,结果表明,序参数S几乎没有发生变化,旋转相关时间τ值的增加证明膜脂过氧化以后,疏水尾部的物理状态发生了改变。经脂质过氧化以后,红细胞膜中的不饱和脂防酸的减少,可能是降低膜脂流动性的原因之一。     

Alterations of erythrocyte structure and cellular susceptibility in patients with chronic renal failure: Effect of haemodialysis and oxidative stress

The aim of this study was to investigate erythrocytes rheological behaviour, membrane dynamics and erythrocytes susceptibility to disintegration upon strong oxidative stress induced by dialysis or by external H₂O₂ among patients with CRF. EPR spectrometry was used to investigate alterations in physical state of cellular components. Generated ROS production induced: (1) significant increase of membrane fluidity in CRF erythrocytes treated with H₂O₂ (p<0.005) and at 60 min of haemodialysis (p<0.05), (2) significant decrease of cytoskeletal protein–protein interactions (p<0.005) and (3) cellular osmotic fragility (p<0.0005). H₂O₂ exacerbated these changes. Erythrocytes from CRF patients have changed rheological behaviour and present higher susceptibility to disintegration. Erythrocytes membrane characteristics indicate that CRF patients possess younger and more flexible cells, which are more susceptible to oxidative stress. This may contribute to the shortened survival of young erythrocytes in CRF patients.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

Comparison of the effect of phenoxyherbicides on human erythrocyte membrane (in vitro)

The molecular mechanisms of phenoxyherbicides action in animals have been insufficiently studied. Now, we have investigated the interaction of sodium salts of phenoxyherbicides, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D-Na), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-Na) and 4-chloro-2-methylphenoxyacetic acid (MCPA-Na) with human erythrocytes. In this study, we evaluated the effect of these compounds on erythrocyte membrane fluidity as well as changes in membrane proteins content. It was observed that all of the compounds studied altered membrane fluidity, changed the size and shape of the erythrocytes and provoked echinocytes formation. It was also revealed that 2,4-D-Na and 2,4,5-T-Na changed the content of erythrocyte membrane proteins mainly by a decrease in the level of spectrin and low molecular weight proteins. The comparison of the action of phenoxyherbicides examined showed that 2,4,5-T-Na caused the greatest changes in the erythrocytes membrane, whereas MCPA-Na induced the lowest alterations in the incubated cells. It must be noted that changes of the investigated parameters were observed only at presence of significant concentrations of these compounds that may penetrate human organism only as a result of acute poisoning.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Alterations of erythrocyte structure and cellular susceptibility in patients with chronic renal failure: effect of haemodialysis and oxidative stress

The aim of this study was to investigate erythrocytes rheological behaviour, membrane dynamics and erythrocytes susceptibility to disintegration upon strong oxidative stress induced by dialysis or by external H(2)O(2) among patients with CRF. EPR spectrometry was used to investigate alterations in physical state of cellular components. Generated ROS production induced: (1) significant increase of membrane fluidity in CRF erythrocytes treated with H(2)O(2) (p<0.005) and at 60 min of haemodialysis (p<0.05), (2) significant decrease of cytoskeletal protein-protein interactions (p<0.005) and (3) cellular osmotic fragility (p<0.0005). H(2)O(2) exacerbated these changes. Erythrocytes from CRF patients have changed rheological behaviour and present higher susceptibility to disintegration. Erythrocytes membrane characteristics indicate that CRF patients possess younger and more flexible cells, which are more susceptible to oxidative stress. This may contribute to the shortened survival of young erythrocytes in CRF patients.     

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r_12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r_12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.     

In vitro effects of the antitumor drug miltefosine on human erythrocytes and molecular models of its membrane

This study was aimed at elucidating the molecular mechanisms of the interaction of the antitumor alkylphospholipid drug miltefosine with human erythrocytes (RBC) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray results showed that the drug interacted with DMPC multilayers; however, no effects on DMPE were detected. The experimental findings obtained by differential scanning calorimetry (DSC) indicated that miltefosine altered the thermotropic behavior of both DMPC and DMPE vesicles. Fluorescence spectroscopy evidenced an increase in the fluidity of DMPC vesicles and human erythrocyte membranes. Scanning electron microscopy (SEM) observations on human erythrocytes showed that miltefosine induced morphological alterations to RBC from its normal biconcave to an echinocyte type of shape. These results confirm that miltefosine interacts with the outer moiety of the human erythrocyte membrane affecting the cell morphology.     

Influence of 2-chlorodeoxyadenosine (cladribine) on human erythrocytes

2-Chlorodeoxyadenosine (2-CdA, cladribine) is one of the newest chemotherapy drugs which has been around and in use for a few years. Drug in tumour cells causes the inhibition of DNA synthesis and repair processes in replication cells, and the accumulation of DNA strand breaks in nonproliferating cells. The present study was undertaken to characterize the influence of cladribine on the fluidity of the lipid bilayer and protein conformation in human erythrocytes. The effect of cladribine on the erythrocyte membrane structure was examined by electron spin resonance (ESR) spectroscopy and fluorescence measurements. It was observed that under the studied conditions (c: 0.1-5 microg/ml, t = 1 h, 37 degrees C), cladribine localised mainly in the erythrocyte membrane and affected its organization. The alterations in the fluidity were observed mainly in the deeper regions of the cell membrane. The incorporation of drug into human erythrocytes also caused negligible conformational alterations of membrane cytoskeletal proteins and did not change the internal viscosity of the cells. We can conclude from these data that 2-CdA in vitro is significantly much less toxic to erythrocytes than anthracycline drugs, which are used in treatment of leukemias. However, the higher concentrations of 2-CdA (about 5 microg/ml) can be also toxic to erythrocytes.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Adiponectin and membrane fluidity of erythrocytes in normotensive and hypertensive men

Objective: Abnormalities in physicochemical properties of the cell membranes may underlie the defects that are strongly linked to hypertension. Recent evidence indicates that adiponectin may have protective effects against cardiovascular diseases. The purpose of the present study was to assess the possible link between plasma adiponectin and membrane fluidity in normotensive (NT) and hypertensive (HT) men. Research Methods and Procedures: We measured the membrane fluidity (a reciprocal value of membrane microviscosity) of erythrocytes in NT and HT men by using an electron paramagnetic resonance and spin‐labeling method. Results: The order parameter (S) for the spin label agent (5‐nitroxide stearate) and the peak height ratio (h₀/h_?1) for 16‐nitroxide stearate in the electron paramagnetic resonance spectra of erythrocytes were significantly higher in HT men than in NT men, indicating that membrane fluidity of erythrocytes was decreased in HT men compared with NT men. Both of plasma adiponectin and nitric oxide (NO) metabolite levels were significantly lower in HT men than in NT men. The plasma adiponectin levels were correlated with plasma NO metabolites. The S and the h₀/h_?1 of erythrocytes were inversely correlated with the plasma adiponectin and NO metabolite levels, indicating that the decreased membrane fluidity of erythrocytes was associated with hypoadiponectinemia and reduced plasma NO metabolites. Discussion: The results of the present study demonstrated that plasma adiponectin levels were lower in HT men than in NT men and that hypoadiponectinemia was associated with decreased membrane fluidity of erythrocytes. The finding suggests that adiponectin may be linked to the rheologic behavior of the erythrocytes and the microcirculation in men, at least in part, by the NO‐dependent mechanism.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Nitric oxide improves membrane fluidity of erythrocytes in essential hypertension: An electron paramagnetic resonance investigation

It has been shown that rheological abnormality might be an etiological factor in hypertension. Recent studies have revealed that human erythrocytes possess a nitric oxide (NO) synthase and that this activation might be involved in the regulation of rheological properties of erythrocytes. The present study was undertaken to investigate the role of NO in the regulation of membrane functions of erythrocytes in patients with essential hypertension by means of an electron paramagnetic resonance (EPR) and spin-labeling method. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(0)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner. The finding indicated that the NO donor increased the membrane fluidity of erythrocytes. In addition, the effect of SNAP was significantly potentiated by 8-bromo-cyclic guanosine monophosphate. By contrast, the change of the fluidity induced by SNAP was reversed in the presence of L-N(G)-nitroarginine methyl ester and asymmetric dimethyl L-arginine. In patients with essential hypertension, the membrane fluidity of erythrocytes was significantly lower than in the normotensive subjects. The effect of SNAP was more pronounced in essential hypertension than in normotensive subjects. These results showed that NO increased the membrane fluidity and decreased the rigidity of cell membranes. Furthermore, the greater effect of NO on the fluidity in essential hypertension suggests that NO might actively participate in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in hypertension.     

The role of intermediates in mitochondrial fatty acid oxidation.

1. Rat liver mitochondria oxidizing [16-14C]palmitoylcarnitine accumulate saturated long-chain thiester intermediates which may be detected by radio-g.1.c.2. Time-courses of intermediate accumulation display no product-precursor relationships and the end product, measured as [14C]citrate, is produced without a detectable initial lag. 3. A short pulse of [16-14C]palmitoylcarnitine followed by unlabelled palmitoylcarnitine showed that the observed intermediates(at least in the greater part)were not the direct precursors of [14C]citrate. 4. The quantity of saturated intermediates depended on the total accumulated flux of acyl units through the pathway provided that some mitochondrial CoA and unused substrate remained. 5. In the presence of rotenone and carnitine, 2-unsaturated, 3-unsaturated and 3-hydroxy intermediates were formed as well as saturated intermediates...     

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.