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1.
Frans A. Kuypers Peter Bütikofer Cedric H.L. Shackleton 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,562(1-2)
We report the application of high-performance liquid chromatographic (HPLC) separation with ultraviolet detection and direct, on-line, structural analyses by mass spectrometry of glycerobenzoate derivatives from complex mixtures of phospholipid molecular species. Individual phospholipids were resolved from total lipid extracts by thin-layer chromatography (TLC). Diradylglycerols were released from phospholipids by phospholipase-C treatment, converted to diradyl glycerobenzoates and subsequently separated by TLC into subclasses (alk-1-enylacyl, alkylacyl and diacyl types). The molecular species within each subclass were resolved by HPLC with an octadecyl reversed-phase column in acetonitrile—isopropanol (80:20, v/v). Individual peaks were quantitated at the picomole level by measuring absorbance at 230 nm. After post-column addition of methanol—0.2 M ammonium acetate (50:50, v/v), peaks were introduced through the thermospray interface into a VG Masslab 30–250 quadrupole mass spectrometer. Molecular species showed as base peaks the salt adducts of the molecular ion which permitted easy deduction of the overall fatty acyl composition. In addition, the diglyceride fragment of each species was found at [MH — 122]+ and two fragments formed by the loss of the fatty acyl groups (R) in the sn-1 or sn-2 position were found at [M — R1]+ and [M — R2]+, respectively. Since preferential release of either fatty acyl group was observed in positional isomers, the ratio of the intensity of these fragments gave information on the position of the fatty acyl groups in the individual HPLC peaks. We show that the use of on-line mass spectrometry, however, provides easy identification of all molecular species present in a complex phospholipid mixture, even when more than one molecular species is contained in an HPLC peak. 相似文献
2.
Svetlana E. Koshlukova Albena B. Momchilova-Pankova Tania T. Markovska Kamen S. Koumanov 《The Journal of membrane biology》1992,127(2):113-119
Summary Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used—palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes—1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A2 which both participate in the deacylation-reacylation cycle. 相似文献
3.
To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells. 相似文献
4.
The fatty acid composition of triglyceride and phospholipids in developing soybean cotyledons (Glycine max L., var. “Harosoy 63”) was analyzed at several stages of growth between 30 and 70 days after flowering. Changes observed in fatty acid composition within each lipid class were related to the levels of lipid molecular species present in the oil. Thirteen molecular species of triglyceride were identified in developing cotyledons, however three of these groups: trilinolenic, dilinolenic-monolinoleic, and linolenic-linoleic-oleic triglycerides, were not found in the mature seed. In immature cotyledons, trioleic and trilinoleic triglycerides accounted for 50% of the structures found; the level of these molecules decreased to 24.9% in the mature seed. The dilinoleic-monolinolenic triglycerides increased from 0.4 to 23.4% during cotyledon development. Changes in triglyceride composition were compared to the levels of molecular species for each phospholipid class. Dilinoleic and monosaturated monolinoleic phospholipid species were dominant in all phospholipid classes throughout development. 相似文献
5.
The incorporation in vivo of 14C-18:2 ω6 and 3H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle. 相似文献
6.
Reaction centres from Rhodopseudomonas sphaeroides, strain R-26, have been solubilised in hexane by the use of phospholipids and cations. Two procedures have been developed: (I) a two-step technique involving the formation of detergent-free proteoliposomes from detergent solubilised reaction centres and phospholipids and mixing these with hexane in the presence of cations; (II) directly sonicating detergent-solubilised reaction centres with phosopholipids before mixing with hexane and cations.The yield of the extracted complex varied with the ratios of protein, phospholipid and cations, species of phospholipid, and sonication time. The spectral characteristics of the complex in the organic phase were similar to those of detergent-solubilised reaction centres. The stability of the reaction centres appeared to be dependent on the presence of phospholipid and water in the hexane. The usefulness of the hexane solution as a model membrane system is discussed and its possible future applications are considered. 相似文献
7.
Mrnka L Nováková O Novák F Tvrzická E Pácha J 《Physiological research / Academia Scientiarum Bohemoslovaca》2000,49(2):197-205
The effect of low-salt diet on phospholipid composition and remodeling was examined in rat colon which represents a mineralocorticoid target tissue. To elucidate this question, male Wistar rats were fed a low-salt diet and drank distilled water (LS, low-salt group) or saline instead of water (HS, high-salt group) for 12 days before the phospholipid concentration and fatty acid composition of isolated colonocytes were examined. The dietary regimens significantly influenced the plasma concentration of aldosterone which was high in LS group and almost zero in HS group. Plasma concentration of corticosterone was unchanged. When expressed in terms of cellular protein content, a significantly higher concentration of phospholipids was found in LS group, with the exception of sphingomyelin (SM) and phosphatidylserine (PS). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for more than 70% of total phospholipids in both groups. A comparison of phospholipid distribution in LS and HS groups demonstrated a higher percentage of PE and a small, but significant, decrease of PC and SM in LS group. The percentage of phosphatidylinositol (PI), PS and cardiolipin (CL) were not affected by mineralocorticoid treatment. With respect to the major phospholipids (PE, PC), a higher level of n-6 polyunsaturated fatty acids (PUFA) and lower levels of monounsaturated fatty acids were detected in PC of LS group. The increase of PUFA predominantly reflected an increase in arachidonic acid by 53%. In comparison to the HS group, oleic acid content was decreased in PC and PE isolated from colonocytes of the LS group. Our data indicate that alterations in phospholipid concentration and metabolism can be detected in rats with secondary hyperaldosteronism. The changes in phospholipid concentration and their fatty acid composition during fully developed effect of low dietary Na+ intake may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function. 相似文献
8.
1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform–methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform–methanol–water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform–methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide. 相似文献
9.
Pang LQ Liang QL Wang YM Ping L Luo GA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,869(1-2):118-125
A rapid and specific analytical method for simultaneous determination and quantification of seven major phospholipid classes in human blood was developed by normal-phase high-performance liquid chromatography tandem mass spectrometry. The optimal separation was achieved by using mobile phase hexane (A) and 2-propanol with water, formic acid and ammonia as modifiers (B) using an HPLC diol column. Isocratic elution method was used for better repeatability and no balance time. The seven major phospholipid classes in human blood that were detected including phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) phosphatidylcholine (PC), lysophosphatidylcholine (Lyso-PC), and sphingomyelin (SM). That can be separated in this condition. Every phospholipid class contains many molecular species which have similar structure. The structure of phospholipids molecular species was identified by ion-trap MS(n) which produced ion fragments. And the qualification was completed by TOF-MS which shows good accuracy. Through the accurate quantification of one representative phospholipids molecule in each class, a method for simultaneous estimation hundreds of molecular species in seven major classes was established. The intra-day and inter-day precision and recovery had been investigated in detail. The RSD of precision for most compound is below 8% and RE is below 10%. Recovery is almost over 80%. This method was applied to phospholipids disorder related with diabetes nephropathy successfully. The concentrations of most phospholipids for normal people are higher than that for diabetic nephropathy (DN) patients in three phases. For most of phospholipids, with the development of DN the concentration was decreasing. 相似文献
10.
Incorporation of radioiodinated fatty acids into cardiac phospholipids of normoxic canine myocardium
G. W. Sloof F. C. Visser T. Teerlink E. F. I. Comans M. J. van Eenige G. J. van der Vusse F. F. Knapp Jr. 《Molecular and cellular biochemistry》1992,116(1-2):79-87
The aim of this study was to assess the phospholipid distribution of radioiodinated 17-iodoheptadecanoic acid (IHDA), 15-(p-iodophenyl)pentadecanoic acid (p-IPPA) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) under normoxic conditions and to compare these data with the fatty acid composition of the phospholipid classes. After simultaneous i.v. injection of the radioiodinated fatty acids (1-123-IHDA; 1-131-p-IPPA; 1-125 DMIPPA) in open-chest dogs seven myocardial biopsies were taken over 40 min (n = 26). After lipid extraction of the biopsies the organic phase was analyzed for both neutral and polar lipids by two different TLC systems. The following polar lipid fractions were analyzed: lysophopshatidylcholine (LPC), sphingomyelin (SPH), phosphatidy1choline (PC; lecithin), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG; cardiolipin) and neutral lipids. Fractions were counted in a gamma well counter and corrected for cross-over and recovery. Results of the polar phospholipids analysis showed that IHDA has the highest incorporation into the phospholipids. The IHDA was mainly incorporated into PI (45.6%) followed by PC (30.9%), PE (14.0%) and PS (5.6%). The p-IPPA was predominantly incorporated incorporated into PC (37.2%), followed by PS (20.1%) and PE (13.7%). In contrast to IHDA, incorporation of p-IPPA into PI was small (6.4%). The DMIPPA analogue was incorporated into phopsholipids to only a very small degree, compared to IHDA and p-IPPA. PS (27.4%) was the only considerable phospholipid fraction into which DMIPPA was incorporated.The results clearly demonstrated that these radioiodinated fatty acid analogues have entirely different patterns of phospholipid incorporation. Major resemblances have been found between the incorporation into phospholipids of IHDA and the phospholipid distribution of the natural counterpart: stearic acid. The p—IPPA phospholipid incorporation only partly resembles the phospholipid distribution of palmitic acid. DMIPPA is because of its modified structure, incorporated into phospholipids to a low extent, mainly into PS. (Mol Cell Biochem116: 79–87, 1992) 相似文献
11.
The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(–20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase—protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion. 相似文献
12.
Jaehun Jung Hyejin Shin Soyoung Bang Hyuck Jun Mok Chang Suk Suh Kwang Pyo Kim Hyunjung Jade Lim 《PloS one》2014,9(7)
Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation. 相似文献
13.
Summary Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques — ruthenium red-osmium tetroxide, osmium tetroxideferrocyanide, acidic phosphotungstic acid in chromate (Rambourg technique) — more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies from — when released — membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium. 相似文献
14.
1. A simple two-phase chloroform–aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of 125I-labelled insulin in this system was determined after incubation at 37°C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized 125I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin–phospholipid or insulin–amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin–phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [14C]glucose between the two phases as a monitor of phospholipid–insulin interactions. The ability of phospholipids to solubilize [14C]glucose in chloroform increased in the order phosphatidylcholine<sphingomyelin<phosphatidylethanolamine<phosphatidic acid. Insulin decreased the [14C]glucose solubilized by phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, but not by sphingomyelin. 5. The significance of these results and the molecular requirements for the formation of insulin–phospholipid complexes in chloroform are discussed. 相似文献
15.
16.
The composition of phospholipids in the liver was studied 7, 14, and 31 days after single oral administration of oil solutions of hexane–ether extracts from potable water sources with different contents of benzo[a]pyrene and polychlorinated biphenyls. Neither linear effect–dose relationship nor directional correlations between individual phospholipid fractions and integrated indices of their composition were revealed, providing evidence for a high sensitivity of phospholipid composition to toxic agents and the absence of its normalization in the mouse liver one month after administration of these agents. 相似文献
17.
Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence 总被引:1,自引:0,他引:1
Microparticles in the circulation activate the coagulation system and may activate the complement system via C-reactive protein upon conversion of membrane phospholipids by phospholipases. We developed a sensitive and reproducible method to determine the phospholipid composition of microparticles. Samples were applied to horizontal, one-dimensional high-performance thin-layer chromatography (HPTLC). Phospholipids were separated on HPTLC by chloroform:ethyl acetate:acetone:isopropanol:ethanol:methanol:water:acetic acid (30:6:6:6:16:28:6:2); visualized by charring with 7.5% Cu-acetate (w/v), 2.5% CuSO(4) (w/v), and 8% H(3)PO(4) (v/v) in water; and quantified by photodensitometric scanning. Erythrocyte membranes were used to validate the HPTLC system. Microparticles were isolated from plasma of healthy individuals (n = 10). On HPTLC, mixtures of (purified) phospholipids, i.e., lysophosphatidylcholine, phosphatidylcholine (PC), sphingomyelin (SM), lysophosphatidylserine, phosphatidylserine, lysophosphatidylethanolamine, phosphatidylethanolamine (PE), and phosphatidylinositol, could be separated and quantified. All phospholipids were detectable in erythrocyte ghosts, and their quantities fell within ranges reported earlier. Quantitation of phospholipids, including extraction, was highly reproducible (CV < 10%). Microparticles contained PC (59%), SM (20.6%), and PE (9.4%), with relatively minor (<5%) quantities of other phospholipids. HPTLC can be used to study the phospholipid composition of cell-derived microparticles and may also be a useful technique for the analysis of other samples that are available only in minor quantities. 相似文献
18.
Jiangwei Yao Philip T. Cherian Matthew W. Frank Charles O. Rock 《The Journal of biological chemistry》2015,290(31):18874-18888
The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine. 相似文献
19.
William Dowhan 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(3):471-494
Although the study of individual phospholipids and their synthesis began in the 1920s first in plants and then mammals, it was not until the early 1960s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960s. In 1970s and 1980s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. 相似文献
20.
Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts. 相似文献