Survival of rabbit and horse erythrocytes in vivo after changing the fatty acyl composition of their phosphatidylcholine

The phospholipid composition and the distribution of phospholipids over the two leaflets of the membrane have been investigated for rabbit and horse erythrocyte membranes. Phosphatidylcholine (PC) comprises 39.4% and 41.3% of the total phospholipid complement of the rabbit and horse erythrocytes, respectively. In both membranes the distribution of this phospholipid is asymmetric: 70% of the PC is present in the outer layer of the rabbit membrane and 60% in that of the horse. The major species of this phospholipid class are the (1-palmitoyl-2-oleoyl)- and the (1-palmitoyl-2-linoleoyl)PC. The disaturated species, (1,2-dipalmitoyl)PC, is present in limited amounts only. Partial replacement of the native PC from intact erythrocytes was accomplished with a purified PC specific transfer protein from bovine liver. Replacement of the native PC species with (1-palmitoyl-2-oleoyl)PC up to 40% of the total PC complement had no effect on the osmotic fragility, the shape and the in vivo survival time of both erythrocyte species. Replacement of the native PC in both rabbit and horse erythrocytes with (1,2-dipalmitoyl)PC up to 20% gave rise to an increased osmotic fragility, a shape change from discocytic to echinocytic and a significant reduction in survival time measured after reinjection of the modified cells. At 30% replacement with (1,2-dipalmitoyl)PC the resulting spheroechinocytes appeared to be cleared from the circulation within 24 h after reinjection. The conclusion can be drawn that the repair mechanisms which may exist in vivo are insufficient to cope with the drastic changes in properties of the erythrocyte membrane which are induced by replacing more than 15% of the native PC by the dipalmitoyl species.     

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

(1) Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. (2) The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. (3) Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K⁺ permeability of the membrane. (4) Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K⁺ permeability was found to be slightly increased in this case. Cells became leaky for K⁺ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. (5) The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine ($\text{unsaturation index}\text{> 0.5}$) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species ($\text{unsaturation index}\text{> 1.0}$).     

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, and a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.Abbreviations LAT lysophosphatidylcholine acyl-CoA transferase - PC phosphatidylcholine - PE phosphatidylethanolamine - PLC propionyl-L-carnitine - STZ streptozotocin     

Erythrocyte insulin receptor characteristics and erythrocyte membrane lipid composition in healthy men

The cell membrane plays an important role in the mechanism of insulin action. To test whether erythrocyte insulin receptor characteristics are related to the erythrocyte membrane lipid composition, 11 healthy volunteers were studied. The relationship between insulin binding to erythrocytes, the number of receptors per cell and the affinity of receptors to insulin on the one hand and total phospholipid fatty acid (FA) composition and cholesterol/phospholipid molar ratio in the erythrocyte membrane on the other hand were evaluated. 1. We found a significant negative correlation between specific insulin binding and the proportion of n-6 essential FA in erythrocyte membrane phospholipids, especially linoleic acid (r = -0.82, p less than 0.01) and arachidonic acid (r = -0.73, p less than 0.05). On the other hand, a significant positive correlation between insulin binding and the proportion of nonessential FA (r = +0.65, p less than 0.05) was seen. Number of receptors per cell and the affinity of receptors were not significantly related to phospholipid FA composition. 2. There was no significant correlation between insulin receptor characteristics and the cholesterol/phospholipid molar ratio in the erythrocyte membrane. The data presented support the hypothesis that the FA pattern of membrane total phospholipids may modify the properties of insulin receptors.     

Lateral diffusion of erythrocyte phospholipids in model membranes comparison between inner and outer leaflet components

The physical properties of lipid bilayers with a similar composition to the outer and inner leaflets of the human erythrocyte membrane have been examined in protein-free model systems. The outer leaflet (OL) was represented by a phospholipid mixture containing phosphatidylcholine and sphingomyelin extracted from human erythrocytes, while a mixture of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine represented the inner leaflet (IL). The ratio of cholesterol to phospholipid was varied in both mixtures. The lateral diffusion coefficient of fluorescent phospholipids diluted in such lipid mixtures was determined by the modulated fringe pattern photobleaching technique. Contrast curves with a single exponential decay, indicative of homogeneous samples, were obtained only for temperatures above 15 °C and for a cholesterol to phospholipid molar ratio below 0.8. The rate of lateral diffusion was approximately five times faster in IL than in OL multilayers, in agreement with former results obtained in human erythrocytes (Morrot et al. 1986). Varying the cholesterol to phospholipid ratio from 0 to 0.8 (mol/mol) enabled us to decrease the diffusion constant by only a factor of approximately 2 for both IL and OL mixtures. The order parameter of a spin-labeled phospholipid was determined in the different systems and found to be systematically smaller in IL mixtures than in OL mixtures. The present study indicates that the difference in lipid diffusivity of the two erythrocyte leaflets may be accounted for solely by a difference in phospholipid composition, and may be independent of cholesterol and protein asymmetry.Abbreviations OL outer leaflet - IL inner leaflet - RBC red blood cell - NBD-PC 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] dodecanoyl phosphatidylcholine - NBD-PE 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] dodecanoyl phosphatidylethanolamine - NBD-PS 1-acyl-2-[12-(7-nitrobenz-2-oxy-1,3-diazol-4-yl)amino] dodecanoyl phosphatidylserine - DMPC 1,2 dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2 dimyristoyl-snglycero-3-phosphoserine - PC phosphatidyleholine - C/P cholesterol over phospholipid molar ratio - D lateral diffusion coefficient - S order parameter - ESR electron spin resonance - NMR nuclear magnetic resonance - EDTA ethylene diamine tetraacetic acid - TRIS tris-(hydroxymethyl)amino ethane Offprint requests to: P. F Devaux     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Modification of the erythrocyte membrane by a non-specific lipid transfer protein

The non-specific phospholipid transfer protein purified from bovine liver has been used to modify the phospholipid content and phospholipid composition of the membrane of intact human erythrocytes. Apart from an exchange of phosphatidylcholine between the red cell and PC-containing vesicles, the protein appeared to facilitate net transfer of phosphatidylcholine from the donor vesicles to the erythrocyte and sphingomyelin transfer in the opposite direction. Phosphatidylcholine transfer was accompanied by an equivalent transfer (on a molar basis) of cholesterol. An increase in phosphatidylcholine content in the erythrocyte membrane from 90 to 282 nmol per 100 microliters packed cells was observed. Phospholipase C treatment of modified cells showed that all of the phosphatidylcholine which was transferred to the erythrocyte was incorporated in the lipid bilayer. The nonspecific lipid transfer protein used here appeared to be a suitable tool to modify lipid content and composition of the erythrocyte membrane, and possible applications of this approach are discussed.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Differential accessibility of bilirubin to erythrocyte membrane vesicles bearing different structural features

Interaction of bilirubin with different types of erythrocyte membrane vesicles such as unsealed, heterogeneous, sealed and inside-out membrane vesicles prepared from human and goat erythrocytes was studied. Out of various types of membrane vesicles, in both species, unsealed membrane vesicles bound quantitatively higher amounts of bilirubin followed by heterogeneous and sealed membrane vesicles whereas inside-out membrane vesicles bound the lowest amount of bilirubin. These differences in the amount of bound bilirubin to different membrane vesicles were correlated well with the percentage accessibility of sialic acid to neuraminidase in these membranes suggesting that bilirubin bound preferentially to the outer layer of erythrocyte membranes than the inner layer. Further, membrane vesicles prepared from human erythrocytes bound higher amounts of bilirubin than those prepared from goat erythrocytes. This can be ascribed to different phospholipid composition of these membranes.     

Modification of the erythrocyte membrane by a non-specific lipid transfer protein

The non-specific phospholipid transfer protein purified from bovine liver has been used to modify the phospholipid content and phospholipid composition of the membrane of intact human erythrocytes. Apart from an exchange of phosphatidylcholine between the red cell and PC-containing vesicles, the protein appeared to facilitate net transfer of phosphatidylcholine from the donor vesicles to the erythrocyte and sphingomyelin transfer in the opposite direction. Phosphatidylcholine transfer was accompanied by an equivalent transfer (on a molar basis) of cholesterol. An increase in phosphatidylcholine content in the erythrocyte membrane from 90 to 282 nmol per 100 μl packed cells was observed. Phospholipase C treatment of modified cells showed that all of the phosphatidylcholine which was transferred to the erythrocyte was incorporated in the lipid bilayer. The nonspecific lipid transfer protein used here appeared to be a suitable tool to modify lipid content and composition of the erythrocyte membrane, and possible applications of this approach are discussed.     

Molecular species composition of membrane phosphatidylcholine influences the rate of cholesterol efflux from human erythrocytes and vesicles of erythrocyte lipid

The efflux of [3H]cholesterol from prelabelled human erythrocytes having modified phosphatidylcholine compositions was measured during 24-h incubations in the presence of unlabelled acceptor liposomes composed of equimolar amounts of egg phosphatidylcholine and cholesterol. The cells were modified by replacement of part of the native phosphatidylcholine with either dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine or dilinoleoylphosphatidylcholine catalyzed by phosphatidylcholine-specific transfer protein from bovine liver. The results indicated that the efflux of [3H]cholesterol was faster from erythrocytes in which the dipalmitoylphosphatidylcholine content was increased from 7 to 25% of the total, than from cells enriched in palmitoyloleoylphosphatidylcholine or dioleoylphosphatidylcholine. Incorporation of dilinoleoylphosphatidylcholine to a level of 13% of the total phosphatidylcholine slowed the rate of efflux of [3H]sterol. The phosphatidylcholine replacements produced no significant differences in cholesterol/phospholipid ratio before or after 24 h of incubation with the acceptor egg phosphatidylcholine-cholesterol vesicles. Using vesicles prepared from erythrocyte lipid, modified to reflect the changes in the phosphatidylcholine composition induced in the whole cells, the same influence of composition on the rate of cholesterol exchange was evident. Enhancement of the dipalmitoylphosphatidylcholine content from 7 to 25% of the total phosphatidylcholine pool increased the rate of [3H]cholesterol efflux, while the addition of the same amount of dilinoleoylphosphatidylcholine slowed it compared to controls. The magnitude of the effect was comparable in intact cells and erythrocyte lipid vesicles enriched in dipalmitoylphosphatidylcholine, while the influence of dilinoleoylphosphatidylcholine was more marked in the intact cells. These results demonstrate that changes in the molecular species composition of the phosphatidylcholine pool can influence the rate of exchange of cholesterol but not necessarily the cellular content of sterol in the human erythrocyte. The influence of this phospholipid appears to be expressed independently of the presence of membrane protein or an underlying cytoskeleton.     

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15–20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.     

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.     

Acyl selectivity in the transfer of molecular species of phosphatidylcholines from human erythrocytes

This report describes the molecular species composition of phosphatidylcholines (PC) transferred from human erythrocytes to acceptor vesicles composed of cholesterol and single PC species in the presence of PC-specific transfer protein from bovine liver. The compositions of the PC isolated from the vesicles were determined by capillary GLC as the diacylglycerol trimethylsilyl ethers. The cellular PC species appearing in the acceptor vesicles were enriched in unsaturated species and showed a low content of dipalmitoyl PC compared to untreated erythrocytes. This trend was independent of the composition of the PC used to construct the acceptor vesicles and it was possible to determine that the relative rates of efflux of the palmitoyl-containing phosphatidylcholines decreased in the order: palmitoyl-linoleoyl greater than palmitoyl-oleoyl greater than dipalmitoyl and in the stearoyl series, stearoyl-linoleoyl greater than stearoyl-oleoyl. No clear trend was distinguished for the influence of chain-length on the efflux, thus preventing an unambiguous assignment of the order of removal of all species from the cell membrane. Results derived for arachidonoyl-containing species were compromised by evidence for oxidation occurring during incubations at 37 degrees C. To confirm that acyl selectivity was also possible during transfer in the absence of the transfer protein, the efflux of 14C-labeled soya PC and [14C]dipalmitoyl PC from prelabeled erythrocytes was measured using plasma as the acceptor. As predicted by the chromatographic analyses, 14C-labeled soya PC effused up to 10-times faster than [14C]dipalmitoyl PC from the red cell membrane. Thus, the more rapid transfer of unsaturated PC cannot be explained entirely as a specificity of the transfer protein and is consistent with the hypothesis that intermolecular interactions involving PC molecules within the erythrocyte membrane, become weaker with increasing unsaturation. The results suggest a potential role of PC-specific transfer protein as a probe of the nature of PC interactions within biological membranes.     

Flip-flop rates of individual molecular species of phosphatidylcholine in the human red cell membrane

Trace amounts of four different, well-defined species of phosphatidyl[N-methyl-14C]choline ([14C]PC), differing in their fatty acyl constituents, were introduced exclusively into the outer membrane leaflet of the intact erythrocyte by using a PC-specific phospholipid transfer protein. The rate of transbilayer equilibration of these probe molecules was calculated from the time-dependent decay in specific radioactivity of the PC pool in the outer monolayer, which was discriminated from that in the inner leaflet by treating the intact cells with phospholipase A2 in the presence of sphingomyelinase C. At 37 degrees C, 1,2-dipalmitoyl-, 1,2-dioleoyl-, 1-palmitoyl-2-linoleoyl- and 1-palmitoyl-2-arachidonoyl-PC revealed halftime values for the rate of their transbilayer equilibration of 26.3 +/- 4.4, 14.4 +/- 3.5, 2.9 +/- 1.7 and 9.7 +/- 1.6 h, respectively.     

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.     

Species-dependent differences in the influence of ionic strength on potassium transport of erythrocytes. The role of membrane fluidity and Ca2+

The passive Rb+ (K+) efflux from erythrocytes of seven mammalian species was investigated in solutions of physiological and low ionic strength. Furthermore the fluidity of the erythrocyte membrane in the same solutions was estimated by measuring the ESR order parameter. The rate constant of Rb+ (K+) efflux in solution of high ionic strength could be correlated with the order parameter obtained and with the mean number of double bonds to the membrane phospholipid fatty acids. The same relationships could be observed for the low ionic strength solutions if the values for human erythrocytes were excluded. The appearance of Na+, K+, Cl- cotransport to a significant extent, only in human erythrocytes, was supposed to be the reason for this different behaviour of human red blood cells. It was demonstrated that the strong increase of the Rb+ (K+) efflux rate constant for human erythrocytes in low ionic strength solution is not due to Ca2+, as quinine treatment and replacement of all external potassium, both inhibiting the Ca2(+)-induced K+ efflux, did not abolish the increase of (Rb+) K+ efflux in solutions of low ionic strength.     

Phospholipid and fatty acid composition of erythrocyte membrane from wild Japanese serow (Capricornis crispus)

The phospholipid composition and fatty acid composition of the individual phospholipids were determined in erythrocyte membrane of wild Japanese serow, Capricornis crispus, and compared with those of Japanese cattle. Sphingomyelin (SM) contributed more than 50% to the total phospholipids, with only 3% phosphatidylcholine, 30% phosphatidylethanolamine and 11% phosphatidylserine. This phospholipid composition and ratio of phospholipid to protein in erythrocyte membrane of wild serow were quite similar to those of Japanese cattle. However, marked differences in fatty acid composition were found, especially in lignoceric acid 24:0 and nervonic acid 24:1 of sphingomyelin which were major constituents (approximately 60%) of that phospholipid.     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.