Electron-spin resonance studies of lipid-modified microsomes from Friend erythroleukemia cells

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5-nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.     

Characterization of highly purified ornithine decarboxylase from rat heart

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5- nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.     

Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells

Improved methods for lipid analysis that have been developed recently were employed to reevaluate the phospholipid composition, the fatty acid and fatty aldehyde composition of the total phospholipid, and the fatty acid composition of the individual phospholipids of normal human red cells. Thirty-three fatty acids and five fatty aldehydes were estimated and tentatively identified in the total phospholipid of normal human red cells. Additional minor components were evident. The major individual phospholipids were isolated by silicic acid thin-layer chromatography and quantified. The fatty acid compositions of phosphatidyl ethanolamine, phosphatidyl serine, lecithin, and sphingomyelin were determined. Each of these phospholipids showed a distinctive and characteristic fatty acid pattern.     

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.     

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.     

Lipid composition of developing Xenopus laevis embryos.

The total lipid content, amount of phospholipid, proportions of major polar and neutral lipid classes, and the overall fatty acid composition were examined in Xenopus laevis embryos. No obvious differences were observed in any of the parameters between fertilization and hatching or between eggs produced by different females. The average lipid content per egg was 113 mug, 31.6 mug of which was phospholipid. The major phospholipids were phosphatidylcholine and sphingomyelin. The major fatty acids were palmitic and oleic acids, but polyunsaturated fatty acids were also present in substantial amounts. The results suggest that significant de novo synthesis of lipids does not occur until after hatching.     

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.     

Evidence for a Composite State of an F′his,gnd Element and a Cryptic Plasmid in a Derivative of Salmonella typhimurium LT2

The zoospores of Blastocladiella emersonii, when derived from cultures grown on solid media, contain about 11% total lipid. This lipid was separated chromatographically on silicic acid into neutral lipid (46.6%), glycolipid (15.8%), and phospholipid (37.6%). Each class was fractionated further on columns of silicic acid, Florisil, or diethylaminoethyl-cellulose, and monitored by thin-layer chromatography. Triglycerides were the major neutral lipids, mono- and diglycosyldiglycerides were the major glycolipids, and phosphatidylcholine and phosphatidylethanolamine were the major phospholipids. Other neutral lipids and phospholipids detected were: hydrocarbons, free fatty acids, free sterols, sterol esters, diglycerides, monoglycerides, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphatidylinositol. Palmitic, palmitoleic, stearic, oleic, gamma-linolenic, and arachidonic acids were the most frequently occurring fatty acids. When B. emersonii was grown in (14)C-labeled liquid media, lipid again accounted for 11% of both mature plants and zoospores released from them. The composition of the lipid extracted from such plants and spores was also the same; however, it differed markedly from that of the lipid in spores harvested from solid media, consisting of 28.3% neutral lipid, 12.0% glycolipid, and 59.7% phospholipid. The major lipids were again triglycerides for neutral lipids, mono- and diglycosyldiglycerides for glycolipids, and phosphatidyl choline and phosphatidylethanolamine for phospholipids.     

Insulin effect on some biochemical and biophysical characteristics of lung surfactant

The incorporation of [14C]palmitic acid into rat alveolar wash total phospholipids and phospholipid fractions has been followed for 6, 8, 10 and 12 hr after insulin administration, indicating a considerable enhancement. The fatty acid profiles of phosphatidylcholines, phosphatidylethanolamines and phosphatidylglycerols were found changed after the hormone administration. Eight hours post insulin treatment the precursor incorporation was highest in all phospholipid fractions studied, as well as the contribution of long chain fatty acids. Dynamic monolayer studies of the lung wash lipid extracts indicated a maximally expanded lipid film corresponding to the highly unsaturated phospholipids present.     

Composition of phospholipids and of phospholipid fatty acids of human plasma

The composition of the phospholipids and of the total phospholipid fatty acids was determined in the plasma of 10 normal subjects. In addition the fatty acid composition of the plasma phosphatidyl ethanolamine, phosphatidyl serine, lecithin, sphingomyelin, and lysolecithin of 6 of the subjects was measured. A wide array of fatty acids was found in the plasma total phospholipid similar to that found previously in red cell total phospholipid. The fatty acid composition in the plasma phospholipids of a given subject reflected that in his red cell phospholipids. Each individual phospholipid displayed a distinctive fatty acid pattern, which was generally similar to that of the corresponding phospholipid of red cells, although some marked differences in individual fatty acid levels between the corresponding phospholipids of plasma and red cells were evident. The high percentage of unsaturated fatty acids found in plasma lysolecithin suggests that this phospholipid did not arise entirely through the enzymatic cleavage of the -fatty acid of lecithin.     

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.     

Use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes

KD115 (ol1), an unsaturated fatty acid auxotroph of S. cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids. The parent strain S288C was studied as a control. The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement. The fatty acid supplement represented 70-80% of the yeast fatty acids. Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1. The mitochondrial morphology of yeast in each growth supplement was notably different. The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures. As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above. The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane.     

Ethynylestradiol alters lipid composition and phosphatidylethanolamine metabolism in red blood cells

Treatment of female rats with ethinylestradiol at a dose of 60 micrograms/rat, daily for 21 days, produced marked changes in red blood cell lipids. Cholesterol was decreased by 22% and total phospholipids were increased by 13%, resulting in a 31% decrease in the cholesterol to phospholipid ratio. The mass distribution of phosphatidylcholine and phosphatidylethanolamine relative to total phospholipids was unchanged. Whereas control red cells incorporated preferentially fatty acids in phosphatidylcholine, ethinylestradiol stimulated their incorporation specifically in phosphatidylethanolamine, where increases occurred with palmitic acid (+75%), oleic acid (+68%) and arachidonic acid (+31%). Incorporation in phosphatidylcholine was unaffected with any of the 3 fatty acids. The stimulation of fatty acid incorporation in phosphatidylethanolamine is likely to reflect an estrogen-dependent increase in turnover rate of fatty acids in this phospholipid. Such alterations in lipid composition and fatty acid incorporation in red cell phospholipids may have significant effects on membrane function.     

Manipulation of Plasma Membrane Fatty Acid Composition of Fetal Rat Brain Cells Grown in a Serum-Free Denned Medium

Modifications of plasma membrane acyl-linked phospholipid fatty acid composition were produced by supplementing the culture medium with essential fatty acids. The plasma membrane fraction was purified by Percoll gradient centrifugation from dissociated fetal rat brain cells grown in a serum-free culture medium. Both the concentration dependence and the time course of the modifications were examined. Supplementation of the medium with essential polyunsaturated fatty acid, linolenic acid (18:3 omega 3) or linoleic acid (18:2 omega 6), produced incorporation of the elongated and desaturated products of omega 3 or omega 6 class, respectively, i.e., the incorporation was class specific. Within each class, the most unsaturated and elongated members, i.e., terminal members, were preferentially incorporated until they reached a maximum concentration within 6-7 days. At higher concentrations of supplemented fatty acids, additional class specific incorporation in plasma membrane was produced by an increase in the concentration of intermediate members. At the same time, the concentration of monounsaturated fatty acids declined and that of saturated fatty acids remained unchanged. The modifications in fatty acid composition were reversible, with the time course similar to that of incorporation. The total plasma membrane phospholipid and sterol contents did not change with alterations of fatty acid composition, but did change with time in culture. This preparation should prove useful for investigating the role of polyunsaturated fatty acids in brain cell functions, including neuronal excitability.     

Phospholipids of Cladosporium resinae cultured on glucose and on n-alkanes.

Cladosporium resinae was grown on glucose, on n-dodecane, and on n-hexadecane. Total lipid was greatest in dodecane-grown cells and least in hexadecane-grown cells, while glucose-grown cells contained the most phospholipid and hexadecane-grown cells contained the least. Cells from all three media contained phosphatidylethanolamine and phosphatidylcholine as their major phospholipids, with lesser amounts of phosphatidylserine and traces of a cardiolipin-like compound. The major fatty acids associated with each phospholipid were palmitic acid and one or more 18-carbon unsaturated fatty acids. There was no correlation between n-alkane growth substrate and fatty acyl components of cellular phospholipids.     

Effect of different levels of thyroid hormones on the fatty acid composition of lipids from rat liver mitochondria

The abundance or deficiency of thyroid hormones in rat organism influence the unsaturation and desaturation indices of total lipid fatty acids and phospholipids in liver mitochondria. The most conspicuous changes were observed in the fatty acid composition of the phospholipid fraction. The changes in the structure and function of rat liver mitochondria are considered to be due to alterations in the fatty acid composition of mitochondrial phospholipids.     

Effect of changes in the membrane lipid composition on the activity of respiratory chain enzymes in rat liver mitochondria

The lipid composition and fatty acid spectrum of individual phospholipid fractions of internal and external membranes of mitochondria was studied in alloxan diabetes. It was found that the phosphatidylserine content is reduced under these conditions, while those of lysophosphatidylcholines, diphosphatidylglycerols and cholesterol are increased, and the fatty acids are saturated with phospholipids. The observed changes in the lipid composition of membranes cause a decrease in the rate of oxygen consumption in various metabolic states as well as in the activity of NAD X H+-, succinate and cytochrome oxidases in rat liver mitochondria.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.