Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Relationship between cell-mediated and humoral immune attack on tumor cells: II. The role of cellular lipid metabolism and cell surface charge in the outcome of immune attack

Treatment of P815 tumor cells with adriamycin increased their sensitivity to killing by anti-P815 antibody plus C, but not by allogeneic P815-sensitized spleen cells. Conversely, mitomycin C treatment enhanced the cells' sensitivity to cell-mediated, but not antibody-C, killing. Hydrocortisone, but not epinephrine, was effective in increasing the resistance of the cells to killing by both antibody-C and cell-mediated attack systems. The ability of the tumor cells to resist antibody-C killing correlated with their ability to incorporate fatty acid into complex cellular lipid; no such correlation was found between the cellular lipid synthesis and tumor cell susceptibility to cell-mediated killing. Drug or hormone-treated tumor cells exhibited unique changes in cellular lipid synthesis and composition and in cell surface physical properties that correlated with their susceptibility to antibody-C or cell-mediated attack. Cells increased in their sensitivity to antibody-C killing exhibited a decreased cholesterol:phospholipid mole ratio. In contrast, cells rendered more sensitive to cell-mediated killing exhibited an increase in polar phospholipid content and a measurable decrease in net negative cell surface charge density. These data implicate unique chemical and/or physical properties of tumor cells to be of fundamental importance for their ability to resist either humoral or cell-mediated immunologic attack; modulation of one or another of these cellular properties results in a change in the cells' susceptibility to immune killing by antibody plus C or by cytotoxic T lymphocytes.     

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

Lipid Composition of Plasma Membranes Isolated from Lactating Bovine Mammary Gland

The light and heavy plasma membranes (PM) isolated from lactating bovine mammary glands contained 38~43% lipid of which 41~44% was phospholipid and 47~52% neutral lipid. The contents of phospholipid and neutral lipid were somewhat higher in the light PM than in the heavy PM. Cholesterol was contained 55 ~60% of neutral lipid and the ratio of cholesterol to phospholipid was 0.64 to 0.69. Phospholipid was composed of sphingomyelin (Sph) 29~38%, phosphatidylcholine (PC) 27~35%, phosphatidylethanolamine (PE) 16~20%, phosphatidylserine 10%, and phosphatidylinositol 6~7%. The content of Sph was higher in the heavy PM than in the light PM, while the values of PC and PE were opposite. The major fatty acids of lipid components were palmitic acid, stearic acid, and oleic acid and those of Sph were palmitic acid, stearic acid, C23:0 and 24:0. The fatty acid composition of individual lipid classes differed significantly from each other but were similar between the light and heavy PMs. Tetracosapentaenoic acid (C24:5) was the major fatty acid of the diacylglycerol fraction. The results indicated that the lipid composition, especially phospholipid components, of bovine mammary gland PMs was different from those of milk fat globule membranes which is derived from the PM of mammary secretory cells.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.     

Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts

The effects sterol carrier protein-2 (SCP-2) expression on L-cell phospholipid levels and fatty acyl composition was assessed using L-cells transfected with the murine cDNA encoding for either the 15 kDa proSCP-2 or 13.2 kDa SCP-2. Expression of these proteins reduced total phospholipid mass (nmol/mg protein) by 24% and reduced the cholesterol to phospholipid ratio 60 and 28%, respectively. In 15 kDa proSCP-2 expressing cells, individual phospholipid class masses, excluding sphingomyelin (CerPCho), were reduced as follows: phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) > ethanolamine glycerophospholipid (EtnGpl) > choline glycerophospholipid (ChoGpl). Furthermore, ethanolamine plasmalogen mass was decreased 25%, while choline plasmalogen mass was elevated 30% in 15 kDa proSCP-2 expressing cells. In 13.2 kDa SCP-2 expressing cells, phospholipid class mass was decreased as follows: PtdIns and PtdSer > ChoGpl. These changes in phospholipid mass resulted in altered cellular phospholipid composition. Expression of either protein differentially altered the type of fatty acid esterified onto the phospholipids. These effects included a greater proportion of polyunsaturated fatty acids and a reduction in saturated fatty acids, although 15 kDa proSCP-2 expression had a more robust effect on these parameters than did 13.2 kDa SCP-2 expression. In summary, expression of SCP-2 reduced individual phospholipid class mass, except for CerPCho, and altered the fatty acid composition of each phospholipid class examined.These results clearly demonstrate that SCP-2 expression altered basal phospholipid levels, suggesting that SCP-2 can alter the function of endoplasmic reticulum phospholipid synthetic enzymes.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Lipid composition and physical properties of membranes from C-6 glial cells with altered phospholipid polar headgroups

Growth of C-6 glial cells in media enriched in the polar headgroup precursors N,N-dimethylethanolamine, N-monomethylethanolamine or ethanolamine for 24 h resulted in the accumulation of the corresponding phospholipids to about 30% of total membrane phospholipid. Under these conditions the cholesterol to phospholipid ratios were unaffected. With the exception of arachidonic acid, which was significantly reduced in the lipids from cells grown in the presence of N-monomethylethanolamine, the fatty acid composition of cells grown under the various conditions was identical. The physical properties of membranes prepared from these cells were compared by electron spin resonance spectroscopy using spin-labelled stearic acid. Modifications in cellular phospholipid composition did not affect either the order parameter or the correlation time of fatty acid spin labels. Since there are no significant effects on the other membrane lipids and since the physical properties of the membranes are maintained, these modifications in phospholipid composition provide a valuable means for studying the role of phospholipid polar headgroups in the function of membrane-bound enzymes and hormone receptors in C-6 cells.     

The partitioning of fatty acid and cholesterol between core and surfaces of phosphatidylcholine-triolein emulsions at pH 7.4

Fatty acids are important intermediate molecules in lipid metabolism. During lipolysis of intracellular lipid droplets or plasma triacylglycerol-rich lipoproteins, fatty acids are generated and may transiently accumulate. We therefore studied the distribution of both fatty acid and free cholesterol between the core and surface of phosphatidylcholine-triolein emulsions at pH 7.4. Nine emulsion systems containing 0.8 to 6.6% cholesterol and 0.16 to 1.02% oleic acid were formed, and core and surface phases were isolated. Phospholipid distributes only to the surface phase. The distribution coefficient of cholesterol surface to core was 23.9 +/- 3.6 S.D., i.e., there was approx. 24-times more cholesterol per unit mass in the surface than in the core phase. This distribution was unchanged by the presence of different quantities of fatty acid in the emulsion particles. The apparent distribution coefficient of fatty acid in surface to core was about 10 at low cholesterol contents and fell to about 7 at high cholesterol contents. However, when the apparent distribution coefficient of fatty acid was related only to the phospholipid component of the surface, the apparent distribution coefficient was constant at about 12.3 +/- 1.1 S.D. Since the fatty acid in the surface phase is about half ionized the true distribution coefficient of unionized fatty acids is about 6.2. The results indicate that fatty acids partition into the phospholipid domains of the surface and not into cholesterol domains and the distribution of fatty acids into surface phospholipid domain is not affected by cholesterol content.     

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.     

Physico-chemical properties of tumor cells that influence their susceptibility to humoral immune killing

Line-10 guinea pig hepatoma cells are susceptible to killing by antibody plus human complement, but resistant to killing by antibody plus guinea pig complement. Tumor cells treated with agents that reversibly increase (adriamycin), decrease (insulin or hydrocortisone), or have no effect (5-fluorouracil) on the susceptibility of the cells to antibody-complement killing were tested for their lipid and fatty acid composition. Hormone-treated (resistant) cells showed an increased total lipid content, an increased cholesterol: phospholipid ratio, and a depressed level of unsaturated fatty acids in the cellular neutral and phospholipids compared to control untreated cells. Adriamycin-treated (sensitive) cells showed exactly the opposite effects. The lipid composition of both hormone- and adriamycin-treated cells returned to control levels when the cells reverted to control levels of susceptibility to antibody-complement killing. 5-fluorouracil-treated cells were indistinguishable from untreated controls in their lipid and fatty acid composition. The chemical composition of the cell, and its effects on the physical properties of the cellular membranes, therefore appears to be fundamental for the ability of these tumor cells to resist humoral immune attack.     

Lipid protein interactions in mitochondria. VIII. Effect of general anesthetics on the mobility of spin labels in lipid vesticles and mitochondrial membranes

We have studied the effect of general anesthetics on the mobility of two stearic acid spin labels (5-doxyl stearic acid and 16-doxyl stearic acid) in bovine heart mitochondria and in phospholipid vesicles made from either mitochondrial lipids or commercial soybean phospholipids. The general anesthetics used include nonpolar compounds (alcohols, halothane, pentrane, diethyl ether, chloroform) and the amphipathic compound, ketamine. All anesthetics tested increase the mobility of the spin labels in phospholipid vesicles to a limited extent up to a concentration where the ESR spectra become those of free spin labels. On the other hand, anesthetics have a pronounced effect on mitochondrial membranes at concentrations as low as those known to produce general anesthesia; the effect is lower near the bilayer surface (5-doxyl stearic acid) and very strong in the bilayer core (16-doxyl stearic acid). The effects of anesthetics are mimicked by the detergent, Triton X-100. We suggest that the discrepancy between the action of anesthetics in mobilizing the spin labels in lipid vesicles and in membranes results from labilization of lipid protein interactions.     

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1–189 and 190–243) and mouse apoA-I (residues 1–186 and 187–240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.     

Characterization of lipid composition in stimulated human lymphocytes by 1H-NMR

Recent in vivo NMR studies have raised interest in the structural changes of cellular lipids during proliferative activity. We investigated the changes in plasma membrane lipid and total cell lipid during mitogenically-stimulated proliferation of human peripheral blood lymphocytes by extraction of lipids and assay by 500 MHz 1H-NMR. Resonances were assigned using one- and two-dimensional spectroscopic techniques, and signals unique to certain species of lipid were identified. Choline and ethanolamine-containing lipids, glycerophospholipid backbones, sphingolipids, cholesterol, plasmalogens and triacylglycerols were readily detected. Resolution of a number of lipid species was not possible, despite the use of high-resolution techniques. NMR values for proliferation-induced changes in the most easily determined parameters, namely the total cholesterol to total phospholipid molar ratio, and phosphatidylcholine, phosphatidylethanolamine and sphingolipid composition, were found to agree with traditional methods. Differences in phospholipid and fatty acid profiles were found between plasma membranes and total cell lipid for resting values and for response to mitogen.     

Electrospray/tandem mass spectrometry for quantitative analysis of lipid remodeling in essential fatty acid deficient mice

A method utilizing electrospray ionization coupled with tandem mass spectrometry was developed as a facile and rapid method to identify and quantify lipid remodeling in vivo. Electrospray/tandem mass spectrometric analyses were performed on lipids isolated from liver tissue and resident peritoneal cells from essential fatty acid sufficient and deficient mice. Essential fatty acid deficiency was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all cellular lipid species. Qualitative and quantitative changes were measured in the phospholipid and cholesterol ester species directly in the chloroform/methanol lipid extract without any prior chromatographic separation. Lipid remodeling in liver and peritoneal cells from essential fatty acid deficient mice was qualitatively similar in cholesterol ester, phosphatidylcholine, and phosphatidylethanolamine. The monoenoic fatty acids palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were increased markedly, whereas all n-6 and n-3 polyunsaturated fatty acids were nearly depleted in phospholipid and cholesterol ester species. The n-9 polyunsaturated fatty acid surrogate, Mead acid (20:3 n-9), substituted for arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) in phospholipid, but not in cholesterol ester, species. Another notable difference was that adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), both metabolites of arachidonic acid, accumulated in phospholipid and cholesterol ester species of peritoneal cells, but not in liver cells, of essential fatty acid sufficient mice. The overall body of data presented illustrates the implementation of electrospray/tandem mass spectrometry as a method for facile and direct quantification of changes in lipid species during lipid metabolic studies.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Effect of ethanol on cholesterol and phospholipid composition of HeLa cells

Chronic exposure of animals to ethanol leads to changes in membrane lipid composition which may be related to the development of tolerance and physical dependence. The object of the present study was to investigate this phenomenon at a cellular level. HeLa cells were grown in the presence of ethanol (86 mM) for periods of up to 9 days. Both the cholesterol and phospholipid concentration of these cells increased during this period but the cholesterol:phospholipid ratio remained unchanged. Among the phospholipid classes phosphatidic acid decreased while phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine increased rapidly, returning toward control values by 9 days. Significant decreases were observed in saturated (14:0, 16:0) and monoenoic (16:1, 18:1) fatty acids while the major polyenoic fatty acid (20:4) increased. It is concluded that cultured mammalian cells represent a useful model for investigation of the direct effects of ethanol on membrane lipid metabolism.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.