Glycerophospholipid Classes Research Articles

Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56lck and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-β-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca2+ concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS 2 or MS 3, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1–5 ng of injected substance.

High dietary levels of a conjugated linoleic acid mixture alter hepatic glycerophospholipid class profile and cholesterol-carrying serum lipoproteins of rats

To investigate the body composition, hepatic lipids, and serum lipoproteins in response to graded levels of a conjugated linoleic acid (CLA) mixture added to a high linoleate diet, adult male Sprague-Dawley rats were randomly assigned into four dietary groups of 10 rats each and fed for 5 weeks controlled amounts of diets containing 0%, 1%, 3%, or 5% of a CLA mixture in exchange for sunflower oil. The various dietary lipid treatments did not significantly influence growth and body partitioning, although there was a trend toward decreased contents of extractable lipids in carcass (whole bled body without liver and gut) with increasing CLA. When carcass lipids of CLA-treated rats were extracted, a distinct accumulation of total CLA was observed. A dietary level of 1% CLA mixture exhibited only weak effects on hepatic glycerophospholipid levels. CLA levels of 3% and 5% caused distinct changes in phospholipid subclass distribution. These changes were reduced levels of lysophosphatidylethanolamine (LPE) and ethanolamine plasmalogen (EPL) and increased levels of phosphatidylcholine (PC). Further, a 5% level of CLA increased the hepatic concentration of phosphatidylserine (PS) compared with the other treatments. The incorporation of total CLA into individual phospholipids followed a dose-responsive manner. The extent of incorporation of CLA was not the same among the glycerophospholipid species analyzed, the order being cardiolipin > phosphatidylethanolamine and PC > LPE/EPL > phosphatidylinositol > PS. Further, CLA increased the proportions of n-3 fatty acids in the individual glycerophospholipids. High CLA diets containing 3% and 5% of a CLA mixture were associated with increased activity of catalase in the peroxisome-enriched cell fraction of liver and exhibited marked reductions of cholesterol in the low and high density lipoproteins relative to rats receiving no CLA.

Fatty acid composition of brain glycerophospholipids in peroxisomal disorders.

This paper shows for the first time the differential fatty acid composition of ethanolamine plasmalogens (EP) and phosphatidylethanolamine (PE) in the brains of 12 patients with disorders of peroxisomal biogenesis and compares the results to normal values for the age. Other important glycerophospholipids (GPL), such as phosphatidylserine (PS) and phosphatidylcholine (PC), are also included in this study. GPL were separated by two-dimensional thin-layer chromatography, and their fatty acid composition was determined by capillary column gas-liquid chromatography. Total brain GPL were slightly decreased in peroxisomal disorders (27.98+/-2.95 micromol/g in the patients against 34.5+/-6.21 micromol/g in age-matched controls, P = 0.005), and the distribution of the different GPL classes was much altered. In confirmation of known data, EP were very much decreased (2.18+/-1.3 micromol/g in the patients against 6.9+/-2.3 micromol/g in controls) at the expense of PE, which was increased (8.58+/-2.17 micromol/g in the patients against 5.97+/-0.58 micromol/g in controls, P<0.005). PS and PC were both significantly decreased (P = 0.0001 and P = 0.037, respectively). The polyunsaturated fatty acid (PUFA) composition of all the GPL fractions was markedly abnormal. In absolute terms, docosahexaenoic acid (22:6n-3) was drastically decreased in all GPL classes (always at the P<0.0001 level) while arachidonic acid (20:4n-6) was increased in PE and PS (P<0.001 in both cases). In the alkenyl acyl form, EP, 22:6n-3, and 20:4n-6 were both very significantly decreased (P<0.0001), although the former was always the most affected. The myelin PUFA adrenic acid (22:4n-6) was decreased in EP (P<0.0001) and slightly increased in PS (P<0.05). The changes found confirm that 22:6n-3 deficiency is a predominant defect in the brain in peroxisomal disorders.

Effects of essential fatty acid-deficient diets on growth, mortality, tissue histopathology and fatty acid compositions in juvenile turbot (Scophthalmus maximus)

Three diets in which the lipid component was supplied either as fish oil (FO), linseed oil (LO) or olive oil (OO) were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 1.2 g for a period of up to 12 weeks. The latter two diets resulted in a significant reduction in specific growth rate and an increased mortality compared to the FO (control) fed fish. A liver histopathology was evident in around half of the fish fed the LO and OO diets but was absent in fish fed FO. The lesion showed indications of cellular alterations consisting of foci of densely basophilic cells but without evidence of inflammatory activity. The total lipid fatty acid composition of the carcass from fish fed LO had increased percentages of 18:2n-6 and 18:3n-3, but decreased percentages of all other polyunsaturated fatty acids (PUFA) including the physiologically important 20:4n-6, 20:5n-3 and 22:6n-3, compared to fish fed FO. Almost 2/3 of the total fatty acids in the carcass of OO-fed fish were monounsaturated while the percentages of total saturated fatty acids and all other PUFA, except 18:2n-6, were significantly reduced compared to fish fed FO. Broadly similar effects on total lipid fatty acid composition were observed in liver. In the liver glycerophospholipid classes of fish fed LO, percentages of 18:2n-6, 18:3n-3 and 20:3n-3 were significantly increased whereas all C20 and C22 PUFA, with the exception of 20:5n-3 in PI, were significantly reduced compared to fish fed FO. The liver glycerophospholipids of fish fed OO all showed significantly increased total monounsaturates, 18:2n-6, 20:2n-6, 18:2n-9 and 20:2n-9 as well as reduced percentages of 20:4n-6 and 22:6n-3, compared to fish fed FO. The brain glycerophospholipids showed broadly similar changes in response to dietary treatment although the magnitude of fatty acid alterations was less than those observed in liver. The greater mortalities in the OO-fed fish compared to the LO-fed fish suggests that incorporation of 18:3n-3 into tissue phospholipids can offset losses of long-chain PUFA more effectively than incorporation of 18:1n-9. However, levels of dietary long-chain PUFA must be optimised to allow normal growth and development. We conclude that the very low flux through the fatty acid desaturase/elongase pathways in turbot is not up-regulated by diets deficient in 20:5n-3 and 22:6n-3.

Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry.

Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.

Lipid structure of rat adipocyte plasma membranes following dietary lard and fish oil

We have determined the changes in the lipid structure of the adipocyte plasma membranes of rats receiving lard or fish oil in their diet. For this purpose, mature Wistar rats were fed 20% (w/w) lard or fish oil diets for 22 days, when the plasma membranes of the epididymal and perirenal adipocytes were prepared. Detailed analysis of the membrane lipids by chromatographic methods showed that dietary fat exerted a major effect on the lipid class and molecular species composition of the phospholipids. As a result of fish oil feeding, significant increases in the 20:5( n−3), 22:5( n−3) and 22:6( n−3) were detected in all glycerophospholipid classes, while the 18:1( n−9) and 18:2( n−6) and to a lesser extent 20:4( n−6) decreased. Incorporation of n−3 fatty acids increased the phosphatidylcholine/sphingomyelin ratio without changing the total phospholipid or free cholesterol content of the membrane. Fish oil feeding also caused a marked increase in the proportion of 24:1 in sphingomyelins, which occurred mainly at the expense of 18:0 and 24:0. New n−3 fatty acid-containing species appeared in the choline and ethanolamine glycerophospholipids, when compared to membrane lipids from lard-fed rats. Membranes from fish oil fed rats also had moderately higher levels of ether lipids. Few differences were seen between the membranes of the epididymal and perirenal adipocytes. It is concluded that dietary fish oils modify the lipid structure of rat adipocyte plasma membranes by increasing the ratio of phosphatidylcholine to sphingomyelin and by increasing the proportion of molecular species with polyunsaturated fatty acids, which would be anticipated to increase the fluidity of the lipid bilayer of adipocyte plasma membranes.

Stimulation of proliferation of an essential fatty acid-deficient fish cell line by C 20 and C 22 polyunsaturated fatty acids and effects on fatty acid composition

Recently we reported the development of a fish cell line, EPC-EFAD, derived from the carp ( Cyprinus carpio) epithelial papilloma line, EPC, that could survive and proliferate in essential fatty acid-deficient (EFAD) medium. The EPC-EFAD cell line may be a useful model system in which to study the cellular biochemical effects of EFA deficiency and has advantages in studies of polyunsaturated fatty acid (PUFA) and eicosanoid metabolism in fish in that the complications introduced by culture in relatively n-6 PUFA-rich mammalian sera are removed. In the present study, the effects on cell proliferation rate of supplementing EPC-EFAD cells with various n-3 and n-6 PUFA were investigated to determine the possible role(s) of PUFA in cell growth and division. The selectivity of incorporation of specific PUFA into individual glycerophospholipid classes and the feasibility of reproducing in vivo fatty acid compositions in vitro were also investigated. Proliferation of the EPC-EFAD cell line was stimulated by arachidonic (20:4 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) fatty acids but not by 18:2 n-6 or 18:3 n-3. The differential effects of PUFA on cellular proliferation may be related to the lack of significant Δ5 desaturase activity in the cells at 22°C and may implicate a role for eicosanoids in the mechanism of stimulation of proliferation. PUFA supplementation increased the cytotoxic effects of longer term culture, an effect that was partly alleviated by inclusion of vitamin E in the culture medium. The cells could generally be supplemented with PUFA to produce cellular fatty acid compositions in vitro that were similar to in vivo compositions.

Sample preparation for the analysis of glycerophospholipids by matrix-assisted positive and negative ion 252Cf plasma desorption time-of-flight mass spectrometry

A new sample preparation technique for the analysis of 13 different classes of glycerophospholipids by positive- and negative-ion 252 Cf plasma desorption time-of-flight mass spectrometry is described. Until recently only the direct deposition and the electrospray deposition technique, both yielding mass spectra of poor quality, were applied. The introduction of the bifunctional small molecule matrix 3-(3-pyridyl) acrylic acid (PAA) as sample matrix brings the advantage of selective analyte adsorption from chloroform solution (in two cases also from pure aqueous solution: phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-diphosphate) onto the matrix surface allowing a novel two-step rinsing procedure. Prior to this rinsing procedure, abundant alkali-cationised molecules and an extensive number of diagnostic fragment ions are observed for these compounds. During the first rinsing step excess of alkali cations is removed by cleaning the matrix surface with up to a few mL of water. After the second rinsing step, where chloroform is used, the intensity of (de)protonated molecular ions and adduct ions with the matrix increases significantly. Most of the fragment ions exhibit a significant decrease in relative abundance or even disappear due to lowering the internal energy of desorbed/ionised molecules by removal of non-adsorbed analyte layers from the matrix surface. In cases where analyte molecules do not form protonated and deprotonated molecular ions ([M + H] + or [M ‐ H] ‐ ), respectively, intense peaks due to adduct ions ([M + PAA + H] + and [M + PAA ‐ H] ‐ ) between matrix (PAA) and analyte (M) molecules are observed. These adduct ions most likely originate via a common mechanism independent of the chemical nature of the glycerophospholipid. Choline-containing phospholipids, which do not form [M‐H] ‐ ions, most likely form ion-pairs with the matrix already in the condensed phase yielding [M + PAA ‐ H] ‐ ions in the negative-ion mass spectra. For the nitrogen-free phospholipids (e.g. phosphatidylinositol), which do not form [M + H] + ions, ion-pair formation between lipid and matrix in the condensed phase is also assumed to be the major process of [M + PAA + H] + ion formation in the postive-ion mode. Applying this sample preparation technique to the analysis of normal phase

Insulin Receptor Processing and Lipid Composition of Erythrocyte Membrane in Patients with Hyperlipidemia.

The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

Effects of salinity on the fatty acid compositions of total lipid and individual glycerophospholipid classes of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture

Cells from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce OPs varying from 300 to 500 mOsm kg(-1) and the direct effects of OP (salinity) on the fatty acid compositions of the main glycerophospholipid classes were determined. The most dramatic effects of salinity on total lipid fatty acids were observed in polyunsaturated fatty acids (PUFA) in TF cells. There was a graded decrease in the percentage of 18:2n-9, and consequently total n-9 PUFA, and concomitantly increased percentages of both total n-3 and n-6 PUFA with increasing salinity. The increased n-3 and n-6 PUFA was due to significantly increased percentages of the major fatty acids in each of these groups, namely 22:6n-3 and 20:4n-6, respectively. The reciprocal changes in n-9 PUFA and n-3/n-6 PUFA in TF cell total lipid resulted in the percentage of total PUFA not being significantly affected by changes in salinity. The graded decrease in 18:2n-9 with increasing salinity in TF cells was observed in all the major glycerophospholipids but especially PE, PI and PS. Increasing salinity resulted in graded increases in the percentages of 22:6n-3 in PE and PS in TF cells. The quantitatively greatest increase in the percentage of n-6 PUFA in TF cells occurred with 20:4n-6 in PC, PE and PL. There were less significant changes in the fatty acid compositions of glycerophospholipids in AS cells. However, the proportion of total n-3 + n-6 PUFA in PE varied reciprocally with the proportion of dimethylacetals in response to salinity. Similar reciprocal changes between fatty acids in response to salinity were also evident in the quantitatively more minor glycerophospholipids PS and Pl. In PS, the percentage of 22:6n-3 was significantly lower at 400 mOsm kg(-1) whereas the proportion of total monoenes was significantly higher at that salinity. A similar inverse relationship between total monoenes and 20:4n-6 (and, to a lesser extent total saturates) in response to salinity was noted in PI. The results show that environmental salinity, without whole-body physiological stimuli, has direct effects on the fatty acid composition of major glycerophospholipid classes in fish cells and that these effects differ in cells from different fish species.

1,25‐Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast‐like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 17 beta-estradiol, or dexamethasone on [1-14C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-14C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-14C]AA was diminished by 96-h treatment with 1,25(OH)2D3 (79 +/- 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 +/- 2% of control, P < 0.05). The 1,25(OH)2D3-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-14C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)2D3 was without effect on either total [1-14C]AA uptake or the specific [1-14C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)2D3 treatment decreased hOB cell uptake of [1-14C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)2D3-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17 beta-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.

Possible compensation of structural and viscotropic properties in hepatic microsomes and erythrocyte membranes of rats with essential fatty acid deficiency.

The effect of essential fatty acid deficiency on the structural and dynamic properties of the lipid matrix of rat liver microsomes and erythrocyte membranes was studied. The rate and range of the rotational mobility of 1,6-diphenyl-1,3,5-hexatriene and 2-, 7-, and 12-(9-anthroyloxy)stearate probes in the native membranes and in lipid vesicles prepared with the total lipid extracts of these membranes were evaluated by using differential polarized phase fluorometry. For the anthroyloxystearate probes, two modes of rotation (in and out of the plane of the aromatic anthracene ring) were partially resolved by measuring at different excitation wavelengths. The fat-free diet produces important changes in the fatty acid composition of the different glycerophospholipid classes without affecting the total double-bond number, the relative contents of cholesterol, phospholipid, and protein, and the glycerophospholipid class distribution. The principal changes, more pronounced in liver microsomes than in erythrocytes, are: an increase in nonessential monoene and triene (18:1n-9 and 20:3n-9) and a decrease in essential diene (18:2n-6) and tetraene (20:4n-6). These changes modify the double-bond distribution as a function of the distance from the interphase toward the bilayer interior, with a significant deficit (15% in erythrocytes and 30% in liver microsomes) in the double-bond density in the intermediate region of the membrane leaflet, corresponding to the carbon number 11-12 of an extended saturated acyl chain, and where the 12-anthroyloxystearate probe is located. In spite of the changes in fatty acid composition and double-bond distribution, with the only exception of a slight increase (about 15%) in the "out of the plane" rotation rate of the 7-(9-anthroyloxy)stearate probe in the erythrocyte lipid vesicles, no other significant change is observed. Thus, the changes in fatty acid composition would take place in such a way that at least the average structural and viscotropic properties of the lipid phase of the membrane, sensed by these probes, would be almost exactly compensated.

Determination of intact molecular species of bovine milk 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine by reversed phase HPLC, a multivariate optimization

The molecular species profile, representing the fatty acid combinations on the glycerol backbone, is the most fundamental description of an intact glycerophospholipid class and provides a key to understanding its functional characteristics. We have developed a powerful RP-HPLC method for the separation of molecular species, including so-called “critical pairs”, of natural mixtures of 1,2-diacyl-sn-glycero-3-phosphocholine 1,2-diacyl-sn-glycero-3-phosphoethanolamine from bovine milk and other natural sources. We have used a novel multivariate development and optimization strategy to locate the region of optimum separation conditions within a prechosen domain of the chromatographic parameter space.

Molecular species composition of glycerophospholipids in rat sciatic nerve and its alteration in streptozotocin-induced diabetes

The molecular species compositon of glycerophospholipid classes in nerves of normal and experimentally diabetic rats was determined, sn-1,2-Diacylglycerol (DAG) moieties of purified phospholipids were liberated enzymatically and analyzed as the benzoate derivatives by high-performance liquid chromatography. The most abundant molecular species in phosphatidylinositol (PI) from normal nerve were 18:0 20:4 (54%) and 16:0 18:1 (17%), whereas in phosphatidylcholine (PC), 16:0 18:1 (52%), 16:0 16:0 (12%) and 18:0 18:1 (11%) predominated. In phosphatidylethanolamine and ethanolamine plasmalogen, 18:1 18:1 , 16:0 18:1 and 18:0 18:1 comprised more than 60% and 75% of the molecular species, respectively. Phosphatidylserine was characterized by a high content of 18:0 18:1 (38%) and a relative abundance of the 18:1 20:0 , 18:1 22:0 and 18:1 24:0 molecular species, which together accounted for over 30% of the total. The molecular species profile of phosphatidic acid did not closely resemble that of any other phospholipids or DAG. In diabetic nerve, the molecular species composition of all diacylphospholipids showed a signficant decline in the content of one or more arachidonoyl-containing molecular species. The largest decline occurred in PC and the least in PI. Except in PC, 16:0 20:4 was more depressed than 18:0 20:4 . In combination with previous analyses of DAG molecular species which showed a similar decline in the content of arachidonoyl-containing molecular species in nerve from experimentally diabetic rats (Zhu, X. and Eichberg, J. (1990) J. Neurochem. 55, 1087–1090), the results suggest that nerve DAG arises largely, but not entirely, from phosphoinositides and that PC could be a signficant precursor, especially in diabetic nerve.

Molecular species of glycerophospholipids and diacylglycerols of cultured SH-SY5Y human neuroblastoma cells.

Total lipid extracts were obtained from SH-SY5Y human neuroblastoma cells grown to confluency in mycoplasma-free 10% fetal calf serum. The major glycerophospholipid classes and free diacylglycerols (DAG) were isolated and quantitated by silicic acid and DEAE-cellulose column and thin-layer chromatography. The choline (CGPL), ethanolamine (EGPL), serine (SGPL), and inositol (IGPL) glycerophospholipids were converted to the corresponding diradylglycerols by phospholipase C. The molecular species of the diradylglycerols were determined by capillary gas-liquid chromatography of the trimethylsilyl or t-butyldimethylsilyl ethers. The CGPL was rich in the oligoenoic species and IGPL was rich in the polyenoic species, especially the 18:0-20:4(n-6). The EGPL contained 30-40% diacyl, 60-64% alkenylacyl, and 1-3% alkylacyl species, which were also rich in polyenoic derivatives. Small amounts of alkenylacyl species were detected also in CGPL. The cellular DAG possessed a molecular species composition halfway between those of the DAG moieties of CGPL and IGPL. The cells grown in the presence of 10% calf serum exhibited great variability in the content of 20:3(n-9) fatty acid, which was found to substitute for the 20:4(n-6) acid in the molecular species with 18:0 in both IGPL and DAG. The 20:3(n-9) was largely absent from the CGPL, but occurred also in EGPL, where it was preferentially associated with 18:0 in the diacyl but with 18:1 in the alkylacyl and alkenylacyl species. The detailed documentation of molecular species of glycerophospholipids of the neuroblastoma cells offers new opportunities for identification of the source of free DAG released in transmembrane signalling.

Diacylglycerol composition and metabolism in peripheral nerve.

The content and molecular species composition of 1,2-diacylglycerol (DAG) in rat sciatic nerve was determined and compared with the molecular species profiles for glycerophospholipid classes in order to gain information concerning the metabolic pathways of DAG formation. The level of DAG in freshly dissected epineurium-free nerve (44 +/- 2 pmol/mg wet weight) was 10-40% of that in other tissues and cultured cells. The predominant DAG molecular species were 18:0/20:4 (30%) and 16:0/18:1 (17%). In comparison with phospholipid molecular species patterns, DAG was characterized by a substantial but lower proportion of the 18:0/20:4 species than was found in phosphoinositides, and a significant fraction of saturated species such as those found in phosphatidylcholine. In nerve from diabetic rats, both the content and arachidonoyl-containing molecular species of DAG were reduced. These species were also decreased in individual glycerophospholipids, except for phosphatidylinositol. The distribution of molecular species in phosphatidic acid (PA) did not resemble that of any other phospholipid. A large rise in DAG content occurred when nerve was incubated in vitro. Molecular species analysis indicated that phosphoinositides were the main source, especially during the initial period. This process was virtually abolished in a Ca(2+)-free medium and probably reflects a response to tissue injury. Evidence was obtained for the isomerization of DAG to 1,3-diacylglycerol during incubation. PA content and molecular species composition of incubated nerve did not change. However, inclusion of propranolol, a PA phosphatase inhibitor, caused a 40% accumulation of PA within 10 min, suggesting that formation of this phospholipid is continuous. These findings support the conclusion that DAG is principally derived from phosphoinositides by phospholipase C hydrolysis, but a minor fraction could be derived from phosphatidylcholine either by the action of phospholipase C or via phospholipase D and PA phosphatase. The metabolic origins of PA appear to be diverse.

Endogenous epoxyeicosatrienoyl-phospholipids. A novel class of cellular glycerolipids containing epoxidized arachidonate moieties.

By chromatographic and mass spectral techniques we document in rat liver the presence of new classes of glycerophospholipids which contain an epoxyeicosatrienoate moiety, esterified to the glycerol-sn-2 position. These novel lipids are formed in vivo from endogenous precursors and under physiological conditions. Chromatographic resolution followed by hydrolysis and regioisomeric analysis showed that they consist of the 8,9-, 11,12-, and 14,15-epoxyeicosatrienoyl derivatives of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Their relative concentrations (micromoles of oxidized lipid/mol of phospholipid) were 70, 85, and 106 for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, respectively. Chiral analysis of the fatty acids at sn-2 revealed an enantioselective preference for 8(S),9(R)-, 11(S),12(R)-, and 14(R),15(S)-epoxyeicosatrienoates in all three lipid classes. The data suggest a multienzyme process initiated by cytochrome P-450 epoxidation of arachidonic acid followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and stereoselective lysolipid acylation. These results provide a molecular basis for a potential physiological role of cytochrome P-450 in the biosynthesis of unique cellular glycerolipids and, consequently, in the control of cell membrane structure and function.