Molecular Species Of Phosphatidylcholine Research Articles

Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24–36 carbon atoms (C24–C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC–ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/ sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24–C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/ z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC–ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.

Analysis of hydrophilic and lipophilic choline compounds in radioresistant and radiosensitive glioblastoma cell lines by HILIC-ESI-MS/MS

A new method based on hydrophilic interaction chromatography-electrospray ionisation-tandem mass spectrometry (HILIC-ESI-MS/MS) coupled to the use of a stable isotope labelled substrate was developed to study the metabolism of choline (Cho) compounds in two human glioblastoma multiform (GBM) cell lines with different responses to ionising radiation. Analysis was performed in the positive ion mode using multiple reaction monitoring. This fast, sensitive and selective method enabled the profiling of both hydrophilic and lipophilic Cho-containing compounds, to analyse specifically different phosphatidylcholine (PtdCho) molecular species, and to measure simultaneously native and labelled Cho metabolites. Radioresistant (SF763) and radiosensitive (SF767) cells were incubated for 8 h with d(9)-Cho. Higher native Cho and phosphocholine (PCho) concentrations and higher uptake of d(9)-Cho and formation of d(9)-PCho were found in the radioresistant cell line. The similar low concentrations of native cytidine 5'-diphosphocholine (CDP-Cho) and d(9)-CDP-Cho in both cell lines show that CDP-Cho is the limiting metabolite in the two models. The turnovers (percentage of each d(9)-Cho compound in its respective pool, i.e. native + labelled) were lower in radioresistant cells for all Cho compounds, suggesting a global PtdCho metabolism more active in radiosensitive cells that could be related to their higher proliferation rate.

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

The Alterations of Erythrocyte Phospholipids in Type 2 Diabetes Observed after Oral High-Fat Meal Loading: The FTIR Spectroscopic and Mass Spectrometric Studies

Little is known about the postprandial remodelling of erythrocytes phospholipids (PLs) in type 2 diabetics (T2DM). Therefore, this study aims to compare the alterations of erythrocyte PLs in T2DM to those of healthy subjects after ingestion of a high-fat meal. Eleven T2DM and ten healthy subjects underwent a high-fat meal loading. Erythrocytes were isolated from blood obtained after fasting and 4 h after the meal. Fourier Transform Infrared (FTIR) spectroscopy was initially used to screen erythrocyte PLs by monitoring C-H stretching vibrations. Phosphatidylcholine (PC) molecular species were further investigated by Liquid Chromatography-Electrospray Ionisation-Mass Spectrometry (LC-ESI-MS). For the control group, FTIR revealed postprandial changes in C-H stretching vibrations, particularly of the olefinic band. These findings were supported by LC-ESI-MS data, showing marked changes in PC molecular species, especially of the PC34:1 (where 34 and 1 mean the summed number of carbons and double bonds, respectively). However, similar changes of those were not apparent in the T2DM group. Our results reveal marked postprandial alterations of erythrocyte PC species in healthy subjects whereas only mild alterations are observed in T2DM. The discrepant effects of high-fat meal loading suggest abnormal PC remodelling in the diabetic erythrocyte that may affect its membrane fluidity and integrity.

Evidence that Norflurazon Affects Chloroplast Lipid Unsaturation in Soybean Leaves (Glycine maxL.)

Norflurazon is a bleaching herbicide known to block carotenoid biosynthesis by inhibiting phytoene desaturase activity. Soybean plants were treated with norflurazon, and we examined the effects on the desaturation of lipid molecular species in leaves using ammonium [1-(14)C] oleate labeling. In monogalactosyldiacylglycerol (MGDG), the main chloroplast lipid, a decrease in 18:3/18:3 molecular species and an increase in its precursors 18:2/18:3 and 18:2/18:2 were observed suggesting that the omega(3) FAD7 desaturase activity in planta was inhibited by norflurazon. The in vitro activity of MGDG synthase was also inhibited by 69%. In contrast, the amount of 18:3/18:3 molecular species of phosphatidylcholine (PC) in the extraplastid compartment increased. The observed increase in in vitro lysoPC-acyltransferase activity and activation of desaturation of [1-(14)C] oleate suggest that extraplastid omega(3)FAD3 desaturase was activated. Analysis of the expression of omega(3) FAD3 and omega(3) FAD7 genes in norflurazon treated plants indicate that omega(3) FAD7 and omega(3) FAD3 desaturases are controlled at the post-transcriptional level.

Elimination of the CDP-ethanolamine Pathway Disrupts Hepatic Lipid Homeostasis

Phosphoethanolamine cytidylyltransferase (ECT) catalyzes the rate-controlling step in a major pathway for the synthesis of phosphatidylethanolamine (PtdEtn). Hepatocyte-specific deletion of the ECT gene in mice resulted in normal appearing animals without overt signs of liver injury or inflammation. The molecular species of PtdEtn in the ECT-deficient livers were significantly altered compared with controls and matched the composition of the phosphatidylserine (PtdSer) pool, illustrating the complete reliance on the PtdSer decarboxylase pathway for PtdEtn synthesis. PtdSer structure was controlled by the substrate specificity of PtdSer synthase that selectively converted phosphatidylcholine molecular species containing stearate paired with a polyunsaturated fatty acid to PtdSer. There was no evidence for fatty acid remodeling of PtdEtn. The elimination of diacylglycerol utilization by the CDP-ethanolamine pathway led to a 10-fold increase in triacylglycerols in the ECT-deficient hepatocytes that became engorged with lipid droplets. Triacylglycerol accumulation was associated with a significant elevation in the expression of the transcription factors and target genes that drive de novo lipogenesis. The absence of the ECT pathway for diacylglycerol utilization at the endoplasmic reticulum triggers increased fatty acid synthesis to support the formation of triacylglycerols leading to liver steatosis.

Identification and kinetics of oxidized compounds from phosphatidylcholine molecular species

Heat-induced oxidative modifications of two phosphatidylcholine molecular species as potential functional food components were evaluated. 1-stearoyl-2-oleoyl- sn-glycero-3-phosphocholine (SOPC) and 1-stearoyl-2-linoleoyl- sn-glycero-3-phosphocholine (SLPC) were chosen as models. The optimal temperature for hydroperoxide formation was determined by MS for each standard: 125 °C for SLPC and 150 °C for SOPC. Oxidation was performed at these temperatures and degradation products were identified using LC–ESI-MS combined to an acid treatment. Kinetics of formation of oxidation products from SOPC and SLPC were monitored over 120 min and curves were drawn for each identified structure. Results showed that native phospholipids rapidly decreased with heat and that oxidation products showed functions, such as hydroxyl, oxo or epoxy groups. Kinetics pointed out that some of these quite stable oxidation products are likely to be found in sizable amounts in processed foods containing phospholipids.

Investigation of C‐reactive protein binding to phosphatidyl choline by CZE and ESI‐Mass analysis

A simple CZE has been developed to investigate the binding reactions between C-reactive protein (CRP) and native as well as Cu(2+) oxidized phosphatidyl choline (PC) in this study. An aqueous-organic solvent system containing 20% sodium phosphate, 70% methanol and 10% acetonitrile was selected as the optimal CZE separation buffer. Native PC showed a broad, higher mobility major peak and a sharp, lower mobility minor peak. Oxidized PC (ox-PC) appeared as two adjacent peaks. PC bound to CRP in the presence of metal ions Ca(2+) and Mg(2+). The absorbance at 214 nm for PC+CRP was about five times of that for PC. Surprisingly, ox-PC interacted much more weakly with CRP. The absorbance at 214 nm for ox-PC+CRP was close to that of ox-PC. Meanwhile, ESI-Mass analysis also suggested that four PC molecular species (C16:0/C18:1, C16:0/C18:2, C18:0/C18:2, C18:0/C18:1) bound with CRP since the ion intensities of PC+CRP were lower than that of PC alone. This study uses both CZE and ESI-Mass analysis to characterize the interaction between CRP and native as well as Cu(2+) oxidized PC, and provides new analytical methods for determining the binding reactions. In addition, it also gives new insight into the molecular interactions between CRP and native as well as oxidized PC.

Imaging of lipid species by MALDI mass spectrometry

Recent developments in MALDI have enabled direct detection of lipids as intact molecular species present within cellular membranes. Abundant lipid-related ions are produced from the direct analysis of thin tissue slices when sequential spectra are acquired across a tissue surface that has been coated with a MALDI matrix. The lipid-derived ions can often be distinguished from other biomolecules because of the significant mass defect that these ions present due to the large number of covalently bound hydrogen atoms in hydrophobic molecules such as lipids. Collisional activation of the molecular ions can be used to determine the lipid family and often structurally define the molecular species. Specific examples in the detection of phospholipids, sphingolipids, and glycerolipids are presented with images of mouse brain and kidney tissue slices. Regional distribution of many different lipid molecular species and Na+ and K+ attachment ions often define anatomical regions within the tissues.

Determination of intramuscular phospholipid classes and molecular species in Gaoyou duck

In this study, intramuscular phospholipid classes and molecular species in Gaoyou duck meat were determined. Classes of phospholipids were identified and quantified by normal phase HPLC combined with UV and evaporative light scattering detectors (ELSD). The main phospholipid classes (phosphatidylcholine and phosphatidylethanolamine) were prepared on a semi-preparative silica gel column by HPLC. Reverse phase HPLC was coupled in parallel with both an ELSD and a mass spectrometry in order to characterise molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The results showed that Gaoyou duck meat had high quantities of PC and PE (64.66% and 28.10% of total phospholipids, respectively). Arachidonic acid was mainly present in PE and formed molecular species containing a saturated fatty acid, such as stearic or myristic acid; however, oleic acid together with palmitic or stearic acid formed the main molecular species in PC. The content of the molecular species with polyunsaturated fatty acids in PE accounted for 98.33%, while that in PC only 46.20%.

Spectrum Normalization Method Using an External Standard in Mass Spectrometric Imaging

In this study, we aimed at establishing a procedure for normalizing mass spectra to improve ion distribution images, i.e., sharply defined tissue edges and increased dynamic range, acquired by mass spectrometric imaging (MSI). A crucial problem in MSI is that variations in ionization efficiencies are observed among spectra, which arises from the heterogeneous matrix-analyte crystallization and sublimation during measurement. Such variation could generate incorrect analyte distribution images. For solving this problem, we performed spectrum normalization using an external standard (ES) compound spiked in the matrix solution. We selected methylcarbamyl platelet-activating factor (C-PAF) (C-16) as the ES compound by considering the following two criteria: (1) no other mass peaks overlap the peak of the ES compound, and (2) the ES compound has sufficient ionization capability on the tissue section, in which numerous biological compounds compete to ionize. For evaluating the optimal concentration of C-PAF, we analyzed ion images of coronal mouse brain sections by MSI using matrix solutions containing different concentrations of C-PAF (0, 0.5, 5, 25, and 50 μg/mL). As a result, we found that the optimal concentration of C-PAF for normalization is 50 μg/mL. Employing the procedure, we show improved ion images of phosphatidylcholine (PC) molecular species in the mouse brain section.

Lipidomic analysis of the retina in a rat model of Smith–Lemli–Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species

Smith-Lemli-Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defect in the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944-induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable with the human disease. We evaluated alterations in the non-sterol lipid components of the retina in this model, compared with age-matched controls, using lipidomic analysis. The levels of 16:0-22:6 and 18:0-22:6 phosphatidylcholine molecular species in retinas were less by > 50% and > 33%, respectively, in rats treated for either 2 or 3 months with AY9944. Relative to controls, AY9944 treatment resulted in > 60% less di-22:6 and > 15% less 18:0-22:6 phosphatidylethanolamine molecular species. The predominant phosphatidylserine (PS) molecular species in control retinas were 18:0-22:6 and di-22:6; notably, AY9944 treatment resulted in > 80% less di-22:6 PS, relative to controls. Remarkably, these changes occurred in the absence of n3 fatty acid deficiency in plasma or liver. Thus, the retinal lipidome is globally altered in the SLOS rat model, relative to control rats, with the most profound changes being less phosphatidylcholine, phosphatidylethanolamine, and PS molecular species containing docosahexaenoic acid (22:6). These findings suggest that SLOS may involve additional metabolic compromise beyond the primary enzymatic defect in the cholesterol pathway.

Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.

Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids.

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.

Incorporation of Newly Synthesized Fatty Acids into Cytosolic Glycerolipids in Pea Leaves Occurs via Acyl Editing

In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.

Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration

BackgroundAlterations to pulmonary surfactant composition have been encountered in the Acute Respiratory Distress Syndrome (ARDS). However, only few data are available regarding the time-course and duration of surfactant changes in ARDS patients, although this information may largely influence the optimum design of clinical trials addressing surfactant replacement therapy. We therefore examined the time-course of surfactant changes in 15 patients with direct ARDS (pneumonia, aspiration) over the first 8 days after onset of mechanical ventilation.MethodsThree consecutive bronchoalveolar lavages (BAL) were performed shortly after intubation (T0), and four days (T1) and eight days (T2) after intubation. Fifteen healthy volunteers served as controls. Phospholipid-to-protein ratio in BAL fluids, phospholipid class profiles, phosphatidylcholine (PC) molecular species, surfactant proteins (SP)-A, -B, -C, -D, and relative content and surface tension properties of large surfactant aggregates (LA) were assessed.ResultsAt T0, a severe and highly significant reduction in SP-A, SP-B and SP-C, the LA fraction, PC and phosphatidylglycerol (PG) percentages, and dipalmitoylation of PC (DPPC) was encountered. Surface activity of the LA fraction was greatly impaired. Over time, significant improvements were encountered especially in view of LA content, DPPC, PG and SP-A, but minimum surface tension of LA was not fully restored (15 mN/m at T2). A highly significant correlation was observed between PaO2/FiO2 and minimum surface tension (r = -0.83; p < 0.001), SP-C (r = 0.64; p < 0.001), and DPPC (r = 0.59; p = 0.003). Outcome analysis revealed that non-survivors had even more unfavourable surfactant properties as compared to survivors.ConclusionWe concluded that a profound impairment of pulmonary surfactant composition and function occurs in the very early stage of the disease and only gradually resolves over time. These observations may explain why former surfactant replacement studies with a short treatment duration failed to improve outcome and may help to establish optimal composition and duration of surfactant administration in future surfactant replacement studies in acute lung injury.

Determination of phospholipid molecular species in pork meat by high performance liquid chromatography–tandem mass spectrometry and evaporative light scattering detection

Normal phase high performance liquid chromatography has been optimized for both evaporative light scattering detection and tandem mass spectrometry in order to characterize the natural phospholipids (PL) (classes and molecular species) of raw and cooked pork meat. The PL fraction included phosphatidylcholine (PC) (42.9% ± 4.5 for raw vs 42.6% ± 8.0 for cooked meat), plasmalogen–phosphatidylethanolamine (pPE) and phosphatidylethanolamine (PE) (26.7% ± 3.1 vs 28.5% ± 2.3), cardiolipin (CL) (8.3% ± 2.9 vs 6.3% ± 0.7), sphingomyelin (Sph) (7.5% ± 0.9 vs 8.3% ± 2.1), phosphatidylinositol (PI) (6.8% ± 0.7 vs 6.5% ± 2.1) phosphatidylserine (PS) (4.9% ± 0.5 vs 4.6% ± 1.4) and lysophosphatidylcholine (LPC) (2.9% ± 1.3 vs 3.3% ± 2.6). Arachidonic acid (absent in Sph) was mainly present in pPE and PI and formed molecular species with a saturated fatty acid, such as stearic (as in PI, PS, PE and PC) or palmitic acid (as in PE and PC), or the respective vinyl ethers in pPE (p18:0 and p16:0); however, in PC, arachidonic acid also formed combinations with oleic and linoleic acid. Palmitic acid formed the most abundant molecular species in PC, but not in CL, PE, PI and PS. Unexpectedly, the cooked pork meat showed an increased content of the molecular species of PI and LPC with more unsaturated fatty acids (18:0/20:4 and 18:2, respectively) with respect to raw meat.

Separation and determination of in vitro oxidized phospholipids by capillary zone electrophoresis

A simple capillary zone electrophoresis (CZE) method was used to determine in vitro oxidized phosphatidyl choline (ox-PC). To optimize the capillary electrophoresis (CE) conditions, organic buffer additives, buffer ionic strength, buffer pH, and applied voltage were examined. The optimal CE separation buffer chosen was an aqueous–organic solvent system containing 10% sodium phosphate buffer (5 mM, pH 7.40), 80% methanol, and 10% acetonitrile. One major peak with a small shoulder was found for phosphatidyl choline (PC), whereas one major peak and a complex region containing several lower-mobility peaks were found for ox-PC. The lower-mobility species of ox-PC has high levels of conjugated dienes characterized by strong absorbance at 234 nm. The electropherograms of PC and ox-PC were significantly different and highly reproducible. The intensities of lower-mobility species decreased significantly when the antioxidant vitamin C concentration was increased from 6 to 600 μM. This study provides a simple CZE method to differentiate in vitro oxidized from nonoxidized PC molecular species.

Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPC(CD36)), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517-38523). A high affinity CD36 recognition motif was defined within oxPC(CD36), an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPC(CD36), a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPC(CD36) precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.