Identification of a potential artifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells

The suitability of electron microscope autoradiography for sutdying the uptake and intracellular localization of lipid vesicles (liposomes) containing radiolabeled saturated phospholipids has been examined. Data are presented showing that preparation of specimens for electron microscope autoradiography by conventional methods is accompanied by significant translocation and intercellular redistribution of radiolabeled saturated lipids, causing spurious labeling patterns. Intercellular redistribution of radiolabeled lipid was demonstrated by mixing glutaraldehyde-fixed mous L1210 cells that had been incubated with sonicated lipid vesicles containing [H] dipalmitoyl phosphatidylcholine with an indicator cell population (fixed avian erythrocytes) which had not been exposed to vesicles and showing that after electron microscope processing radiolabeled grains were present in both cell types. The same redistribution artifact also probably affects the intracellular localization of radiolabeled lipids. This artifact is discussed in relation to previous work in which autoradiographic methods have been used for ultrastructural localization saturated phospholipids in cells and tissues.     

A model for the asymmetric lipid distribution in the human erythrocyte membrane

The asymmetric transverse distribution of phospholipids in the human erythrocyte membrane can be explained by differences between the rate constants of flip and flop motion of the lipids. A selective interaction between aminophospholipids and spectrin does not need to be assumed for creating and maintaining the asymmetric localization of these lipids. Shape transformation of red cells could be caused by alterations of the flip-flop rate constants leading to a change of the lipid distribution and, consequently, to a differential area expansion of the outer and inner membrane leaflet.     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.     

Effect of "marine" phospholipids omega-3 fatty acids on the composition of fatty acids of rat liver microsomal membrane under oxidative stress

As a result of experiments conducted the marine phospholipids preparation enriched by omega-3 fatty acids was defined to modify fatty acids content due to changes of fatty acids level change in the neutral lipids and phospholipids fractions. As well it was identified, that at the oxidative stress induced by administration of CCl4 the growth of arachidonic and docozahexaenoic acids in the neutral lipids fractions was observed if compare with the norm. At the same time, the presented fatty acids in the phospholipids fractions remained unchanged. At oxidative stress the phospholipids fraction reacts to levels of arachidonic and docozahexaenoic acids just only as a result of administrating phospholipids with omega-3 fatty acids. The most attractive is the change of correlation C20:4/C22:6--increasing at administration of CCl4 and decreasing both at phospolipids and vitamin E injection. Thus, at the oxidative stress the first reacting ones are the fatty acids of neutral lipids microsomal membranes.     

Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography–mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.     

Lipids at the stages of early osteogenesis in human long tubular bones

In 240 long tubular bone anlagen (LTBA) of extremities in 40 human embryos and prefetuses at the age of 6-12 weeks of the prenatal development the investigation has been performed concerning localization, dynamics of contents and spectrum of neutral lipids and phospholipids. Lipids are stained with Sudan III and IV, phospholipids-with Sudan black with corresponding control extractive methods and quantitative estimation of these substances values. The material for electron microscopical investigation of the lipids is treated according to the method, that preserves their safety. The spectrum of lipids and phospholipids in the LTBA is studied by means of the thin layered chromatography method. The data of the histochemical investigation and chromatography demonstrate decreasing contents of neutral lipids with the gradient from the zone of the preserved cartilage up to the ossification zone of the epiphyseal cartilage of the LTBA in the human embryos and prefetuses. Increased concentration of phospholipids and complication of their spectrum is noted in the areas of intensive deposits of calcium salts. An essential role of the substance of lipid origin is supposed in ossification and mineralization of the human LTBA.     

Preferential utilization of ketone bodies in the brain and lung of newborn rats

Persistent mild hyperketonemia is a common finding in neonatal rats and human newborns, but the physiological significance of elevated plasma ketone concentrations remains poorly understood. Recent advances in ketone metabolism clearly indicate that these compounds serve as an indispensable source of energy for extrahepatic tissues, especially the brain and lung of developing rats. Another important function of ketone bodies is to provide acetoacetyl-CoA and acetyl-CoA for synthesis of cholesterol, fatty acids, and complex lipids. During the early postnatal period, acetoacetate (AcAc) and beta-hydroxybutyrate are preferred over glucose as substrates for synthesis of phospholipids and sphingolipids in accord with requirements for brain growth and myelination. Thus, during the first 2 wk of postnatal development, when the accumulation of cholesterol and phospholipids accelerates, the proportion of ketone bodies incorporated into these lipids increases. On the other hand, an increased proportion of ketone bodies is utilized for cerebroside synthesis during the period of active myelination. In the lung, AcAc serves better than glucose as a precursor for the synthesis of lung phospholipids. The synthesized lipids, particularly dipalmityl phosphatidylcholine, are incorporated into surfactant, and thus have a potential role in supplying adequate surfactant lipids to maintain lung function during the early days of life. Our studies further demonstrate that ketone bodies and glucose could play complementary roles in the synthesis of lung lipids by providing fatty acid and glycerol moieties of phospholipids, respectively. The preferential selection of AcAc for lipid synthesis in brain, as well as lung, stems in part from the active cytoplasmic pathway for generation of acetyl-CoA and acetoacetyl-CoA from the ketone via the actions of cytoplasmic acetoacetyl-CoA synthetase and thiolase.     

Chronic Alcohol Exposure Increases Circulating Bioactive Oxidized Phospholipids

Ethanol metabolism by liver generates short lived reactive oxygen species that damage liver but also affects distal organs through unknown mechanisms. We hypothesized that dissemination of liver oxidative stress proceeds through release of biologically active oxidized lipids to the circulation. We searched for these by tandem mass spectrometry in plasma of rats fed a Lieber-DeCarli ethanol diet or in patients with established alcoholic liver inflammation, steatohepatitis. We found a severalfold increase in plasma peroxidized phosphatidylcholines, inflammatory and pro-apoptotic oxidatively truncated phospholipids, and platelet-activating factor, a remarkably potent and pleiotropic inflammatory mediator, in rats chronically ingesting ethanol. Circulating peroxidized phospholipids also increased in humans with established steatohepatitis. However, reactive oxygen species generated by liver ethanol catabolism were not directly responsible for circulating oxidized phospholipids because the delayed appearance of these lipids did not correlate with ethanol exposure, hepatic oxidative insult, nor plasma alanine transaminase marking hepatocyte damage. Rather, circulating oxidized lipids correlated with steatohepatitis and tumor necrosis factor-α deposition in liver. The organic osmolyte 2-aminoethylsulfonic acid (taurine), which reduces liver endoplasmic reticulum stress and inflammation, even though it is not an antioxidant, abolished liver damage and the increase in circulating oxidized phospholipids. Thus, circulating oxidized phospholipids are markers of developing steatohepatitis temporally distinct from oxidant stress associated with hepatic ethanol catabolism. Previously, circulating markers of the critical transition to pathologic steatohepatitis were unknown. Circulating oxidatively truncated phospholipids are pro-inflammatory and pro-apoptotic mediators with the potential to systemically distribute the effect of chronic ethanol exposure. Suppressing hepatic inflammation, not ethanol catabolism, reduces circulating inflammatory and apoptotic agonists.     

Developmental differences in activation of cholinephosphate cytidylyltransferase by lipids in rabbit lung cytosol

Lung cytosolic cholinephosphate cytidylyltransferase is activated by lipids. We examined the lipid activation pattern as a function of development in rabbit lung from 27 days gestation through term (31 days) and in the adult. The enzyme in both the fetal and adult cytosol was dependent on lipids for activity. Extraction of the cytosol with acetone/butanol virtually abolished cytidylyltransferase activity, but the activity could be restored on addition of lipids extracted with chloroform/methanol from additional cytosol. Cytosolic phospholipids from the fetal lung reactivated cytidylyltransferase but both neutral lipids and phospholipids from the adult were required. The lipids had the same effect on cytidylyltransferase activity in delipidated cytosol from either the fetus or adult so the difference in activation pattern was attributable to the lipids rather than the protein. There was a shift from the fetal to the adult lipid activation pattern as development progressed. Further, there was a significant correlation between cytidylyltransferase activities in intact cytosols from developing lung and activities in delipidated cytosol in the presence of lipids from the same animals. Although these data suggest that lipids regulate cytosolic cytidylyltransferase activity in developing lung their physiological significance remains to be established.     

Identification of a potential artifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells

The suitability of electron microscope autoradiography for studying the uptake and intracellular localization of lipid vesicles (liposomes) containing radiolabeled saturated phospholipids has been examined. Data are presented showing that preparation of specimens for electron microscope autoradiography by conventional methods is accompanied by significant translocation and intercellular redistribution of radiolabeled saturated lipids, causing spurious labeling patterns. Intercellular redistribution of radiolabeled lipid was demonstrated by mixing glutaraldehyde-fixed mouse L1210 cells that had been incubated with sonicated lipid vesicles containing [³H]dipalmitoyl phosphatidylcholine with an indicator cell population (fixed avian erythrocytes) which had not been exposed to vesicles and showing that after electron microscope processing radiolabeled grains were present in both cell types. The same redistribution artifact also probably affects the intracellular localization of radiolabeled lipids. This artifact is discussed in relation to previous work in which autoradiographic methods have been used for ultrastructural localization of saturated phospholipids in cells and tissues.     

Alterations in wheat pollen lipidome during high day and night temperature stress

Understanding the adaptive changes in wheat pollen lipidome under high temperature (HT) stress is critical to improving seed set and developing HT tolerant wheat varieties. We measured 89 pollen lipid species under optimum and high day and/or night temperatures using electrospray ionization‐tandem mass spectrometry in wheat plants. The pollen lipidome had a distinct composition compared with that of leaves. Unlike in leaves, 34:3 and 36:6 species dominated the composition of extraplastidic phospholipids in pollen under optimum and HT conditions. The most HT‐responsive lipids were extraplastidic phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidic acid, and phosphatidylserine. The unsaturation levels of the extraplastidic phospholipids decreased through the decreases in the levels of 18:3 and increases in the levels of 16:0, 18:0, 18:1, and 18:2 acyl chains. PC and PE were negatively correlated. Higher PC:PE at HT indicated possible PE‐to‐PC conversion, lower PE formation, or increased PE degradation, relative to PC. Correlation analysis revealed lipids experiencing coordinated metabolism under HT and confirmed the HT responsiveness of extraplastidic phospholipids. Comparison of the present results on wheat pollen with results of our previous research on wheat leaves suggests that similar lipid changes contribute to HT adaptation in both leaves and pollen, though the lipidomes have inherently distinct compositions.     

Lipids of the zygomycete Absidia corymbifera F-965

The cell lipids of the zygomycete Absidia corymbifera F-965 extracted with isopropanol and CHCl3-MeOH mixtures at the exponential growth phase comprise 20+/-2% of mycelium dry wt. The lipids consist of: triacylglycerols (51% of the total lipids extracted), diacylglycerols (9%), monoacylglycerols (3%), ergosterol (5%), ergosterol peroxide (5alpha,8alpha-epidioxyergosta-6,22-diene-3beta-ol) (3%), fatty-acid esters of ergosterol (less than 0.5%), free fatty acids (4%), glucocerebroside (3%), and glycerophospholipids (22%). The main phospholipids are phosphatidylethanolamine (39% of the total phospholipids), phosphatidyl-myo-inositol (17%), diphosphatidylglycerol (12%), phosphatidic acid (7%), phosphatidylcholine (6%), phosphatidylglycerol (3%), and two unusual phospholipids reported earlier, N-acetylphosphatidylethanolamine (7%) and N-ethoxycarbonyl phosphatidylethanolamine (9%). In addition, two unknown acidic phospholipids are present in traces. Saturated fatty acids of the lipids are dominated by n-hexadecanoic acid and unsaturated ones by octadecenoic acid; octadecadienoic and octadecatrienoic acids are present in lesser amounts. Ergosterol peroxide as well as the above glucocerebroside which contains 9-methylsphinga-4(E),9(E)-dienine have first been revealed in zygomycetes.     

Lipid composition of the pea aphid,Acyrthosiphon pisum (Harris) (homoptera: Aphididae), reared on host plant and on artificial media

Polar and neutral lipids and their constitutive fatty acids were quantified in the pea aphid, Acyrthosiphon pisum (Harris), grown on host plant or on a lipid free artificial diet. The results were compared to determine if lipids were involved in the suitability of the diet for continuous rearing of this A. pisum biotype. For apterous adults grown on plants, the lipids were characterized by a low amount of neutral lipids (2.5% weight/fresh weight) almost entirely (96.4%) composed of hexanoyl and sorboyl dimyristin. These storage lipids were higher in the alatae (3.8%), probably correlated with potential flight activity. The phospholipid amounts were identical in these two morphs (1.3–1.4% weight/fresh weight), comprised mainly of phosphatidylethanolamines (52%) and phosphatidylcholines (40.6%). These phospholipids contained a still unidentified fatty acid, with a retention time close to that of linolenic acid and synthesized by the aphid or its bacterial symbionts (not found in plants). The apterous adult aphids reared on an artificial diet showed an accumulation of neutral lipids (8.9% for the first generation); this increase was shown to be slightly greater for the hexanoyl and sorboyl triglycerides. In contrast, the phospholipids decreased in aphids reared on an artificial diet (1.1% and 0.9%, respectively, for first and second generation), correlated with a phospholipid fatty acid profile significantly deficient in C18:3 and in the unidentified peculiar fatty acid. These phospholipids are essential components of biological membranes and a diet-driven deficient synthesis in some of their components may result in the observed symptoms. © 1992 Wiley-Liss, Inc.     

Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases

The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids.     

LIPIDS OF ACANTHAMOEBA CASTELLANII : Composition and Effects of Phagocytosis on Incorporation of Radioactive Precursors

The lipids of Acanthamoeba castellanii (Neff) consist of 52% neutral lipids and 48% polar lipids. Triglycerides account for 75% and free sterols for 17% of the neutral lipids. The major phospholipids are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), a phosphoinositide (6%), and diphosphatidylglycerol (4%). The phosphoinositide is unique in that it contains fatty acids, aldehyde, inositol, and phosphate in the ratio of 1.4:0.5:1.1, but it contains no glycerol. Sphingomyelin, cerebrosides, psychosine, and glycoglycerides were not detected, but small amounts of unidentified long chain bases and sugars are present. The rates of uptake of palmitate-1-¹⁴C and of its incorporation into glycerides and phospholipids were not affected by the phagocytosis of polystyrene latex beads. Although phagocytosis usually decreased the uptake by amebas of phosphate-³²P, serine-U-¹⁴C, and inositol-2-³H, their subsequent incroporation into phospholipids was not demonstrably stimulated or inhibited by phagocytosis. Phagocytosis did seem to increase the incorporation into ameba phospholipids of phosphatidylcholine-1 ,2-¹⁴C but not that of phosphatidylethanolamine-1 ,2-¹⁴C. These experiments, in which the incorporation of radioactive precursors into total cell lipids was measured, do not, of course, eliminate the possibility that localized effects may occur.     

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.     

Synthesis and Development of Starch Granules in High Lysine Barley Grains

Electron microscopy studies of developing endosperm have shown differences in the synthesis and development of starch granules between high lysine mutant Notch-2 and parent NP 113. The starch granules in Notch-2 were modified and did not develop into characteristic oval granules. Based on iodine absorption and phospholipids analysis, it is suggested that the presence of phospholipids impairs the development of starch granule in Notch-2. This is further confirmed by higher lipid density across starch granule in mutant Notch-2, and its absence in NP 113. Amylopectin from mutant differs from that of NP 113. The results indicate that the lack of geometry and smaller size of starch granules in Notch-2 is ultimately due to specific interaction of lipids with developing starch granules, and this leads to decreased yield.     

Changes of Linoleic,Arachidonic and Eicosapentaenoic Acids in Rat Platelet,Aorta and Plasma Lipids after Changing from a Sardine Oil Diet to a Corn Oil Diet

When rats were fed on a diet containing sardine oil (SO) for 10 days, 20:5 (eicosapentaenoic acid) was incorporated into the platelet, aorta and plasma lipids. The 20:5/20:4 (arachidonic acid) ratios of phospholipids were higher in platelets than in aortas. When the diet was changed from SO to corn oil (CO), the levels of 20:5 in the plasma lipids and platelet phospholipids decreased with an increase of 20:4. Although the 20:5 levels of phospholipids in the aortas decreased, 20:4 did not increase during feeding with the CO diet for 4 days. The 20:5/20:4 ratios of the phosphatidylcholine in platelets and aortas rapidly changed from 5.0 and 1.1 to 2.4 and 0.6, respectively. The linoleic acid level of phosphatidylcholine in platelets and aortas increased rapidly by changing from the SO diet to the CO diet, but this fatty acid was not incorporated into phosphatidylinositol of the platelets and aortas.     

Isolation of the membranes of Actinomyces sp. 26-115, a producer of actinomycin C]

A summation fraction of the membranes of Actinomyces sp. 26-115 was obtained as a result of lysis of its protoplasts in a hypotonic medium. The qualitative content of protein, lipids, phospholipids, nucleic acids, glucosamine and muramic acid was determined in the membranes at various stages of the organism development. Phosphatidylcholine is the main component of phospholipids in this organism. Intracellular actinomycin was found inside the protoplasts. Electrophoregrams of the microprotoplasts and membranes are presented. Actinomycin was also detected in the membranes. Still, it is not clear whether it is a component of the membrane or it is adsorbed on the membrane during the process of its isolation. The final conclusion on the relationship between the membrane and localization of actinomycin in the cell requires further investigation.     

Polyunsaturated fatty acids in serum lipids of reindeer during the close postnatal period

We examined serum fatty acid composition in reindeer during the close postnatal period (from <8 h to 3 weeks) by using maternal serum as a reference point and focusing on the proportions of polyunsaturated fatty acids (PUFAs) in serum lipids. A striking dissimilarity was found in the serum PUFAs between the neonatals and their mothers. In particular, the proportions of linoleic acid (18:2) and α-linolenic acid in serum cholesteryl esters and phospholipids of the newborn reindeer were significantly lower than those of the mothers. Furthermore, serum phospholipids of the newborns had lower arachidonic acid and docosapentaenoic acid but higher docosahexaenoic acid proportions than the maternal phospholipids. Although the proportions of the principal C18 PUFAs were low in reindeer milk, they increased sharply in serum cholesteryl esters and phospholipids of the calves during the first few days after birth. In particular, there were significant positive correlations in the proportions of 18:2 between serum and milk lipids. We conclude that the proportions of the serum C18 PUFAs are low in the newborn reindeer, but they are increased during the close perinatal period by a rate which suggests an efficient mechanism for selective retention of these fatty acids from milk lipids. Accepted: 10 August 1998