Molecular Species Of Phosphatidylcholine Research Articles

Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein

BackgroundOxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.MethodsHuman and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry.ResultsWhen human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL.ConclusionsThese results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.

Combination of metabolomic and phospholipid-profiling approaches for the study of Alzheimer's disease

Alzheimer's disease is closely related to abnormal metabolism of phospholipids from neural membranes, so that the study of their dyshomeostasis could be of great interest for the discovery of potential biomarkers for diagnosis and disease monitoring. In this work, it has been developed a metabolomic multi-platform for the characterization of phospholipid alterations occurring in serum from Alzheimer's disease patients. For this purpose, we performed a metabolomic screening by direct infusion mass spectrometry and profiling analysis by reversed phase ultra-high performance liquid chromatography with complementary detection by molecular and atomic mass spectrometry, which allowed combining the high-throughput capability of shotgun metabolomics and the targeted character of profiling approaches. Thus significant changes were detected in the levels of several molecular species of phosphatidylcholines, phosphatidylethanolamines, plasmenylcholines, plasmenylethanolamines and different classes of lysophospholipids, which provided a global vision of the possible factors triggering membrane breakdown. In this sense, alterations of phospholipids metabolism appears to have a multifactorial origin involving overactivation of phospholipases, increased anabolism of lysophospholipids, peroxisomal dysfunction, imbalances in the levels of saturated/unsaturated fatty acids contained in the structure of phospholipids and oxidative stress. This work represents the first comprehensive characterization of serum phospholipids alterations in relation to Alzheimer's disease, by combining shotgun metabolomics and phospholipids profiling through different analytical approaches.This article is part of a Special Issue entitled: Environmental and structural proteomics.

Identification of volatiles from oxidised phosphatidylcholine molecular species using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC-MS)

Headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry analysis (GC-MS) was used to investigate the volatile compounds from oxidised phosphatidylcholine molecular species. 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) were chosen as models. The influence of several parameters on the efficiency of volatile oxidised compounds (VOCs) microextraction, such as type of fibre, extraction duration and temperature were studied. The best results were obtained with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre used at 50 °C during 25 min. The effect of oxidation temperature on the yield of VOCs from SOPC and SLPC was investigated. Oxidative kinetics of SOPC and SLPC were investigated by measuring both the production of VOCs and the degradation of starting materials. More than 30 VOCs were detected by means of the reference mass spectra of the National Institute of Standards and Technology mass spectral library, and most of them were further confirmed by comparing their mass spectra and retention time with those obtained from authentic reference compounds under the same analytical conditions. Moreover, the origins of VOCs from oxidised PLs were studied by comparing those obtained from their corresponding triacylglycerides under the same experimental conditions. The main VOCs identified from oxidised SOPC were (E)-2-decenal, nonanal and octanal and from oxidised SLPC were (E)-2-heptenal, (E)-2-octenal and (E, E)-2,4-decadienal. The proposed method was applied to a real food sample, soy lecithin.

Variability in Associations of Phosphatidylcholine Molecular Species with Metabolic Syndrome in Mexican–American Families

Plasma lipidomic studies using high performance liquid chromatography and mass spectroscopy offer detailed insights into metabolic processes. Taking the example of the most abundant plasma lipid class (phosphatidylcholines) we used the rich phenotypic and lipidomic data from the ongoing San Antonio Family Heart Study of large extended Mexican-American families to assess the variability of association of the plasma phosphatidylcholine species with metabolic syndrome. Using robust statistical analytical methods, our study made two important observations. First, there was a wide variability in the association of phosphatidylcholine species with risk measures of metabolic syndrome. Phosphatidylcholine 40:7 was associated with a low risk while phosphatidylcholines 32:1 and 38:3 were associated with a high risk of metabolic syndrome. Second, all the odd chain phosphatidylcholines were associated with a reduced risk of metabolic syndrome implying that phosphatidylcholines derived from dairy products might be beneficial against metabolic syndrome. Our results demonstrate the value of lipid species-specific information provided by the upcoming array of lipidomic studies and open potential avenues for prevention and control of metabolic syndrome in high prevalence settings.

リン脂質によるシグナル伝達の研究の進歩 －細胞間と細胞内におけるシグナル伝達分子としてのリン脂質分解産物、その代謝物および特定ホスファチジルコリン分子種－

リン脂質によるシグナル伝達の研究の進歩 －細胞間と細胞内におけるシグナル伝達分子としてのリン脂質分解産物、その代謝物および特定ホスファチジルコリン分子種－

S18 Bronchoalveolar Lavage, Tracheal Wash and Induced Sputum Surfactant Phospholipid Kinetics from Healthy Volunteers

Introduction and AimsPulmonary surfactant is a complex mixture of lipoproteins synthesised and secreted by alveolar type II cells. The assessment of surfactant synthetic function and metabolism may provide essential information...

Identification of the Fatty Acyl Residues Composition and Molecular Species of Phosphatidylcholines in Soy Lecithin Powder by UPLC–ESI-MS/MS

The fatty acyl residues composition and molecular species of phosphatidylcholines (PCs) in soy lecithin powder were studied and characterized using ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC–ESI-MS/MS) via a two-step procedure. ESI-MS/MS by precursor ion scan (PIS) m/z 184.1 in positive ion mode first detected isobaric PCs, and further identified positional isomers and fatty acyl residue composition in negative ion mode. The results showed that the fatty acyl residue composition was in strong agreement with that detected by the classical analysis method. Likewise, ten peaks of isobaric PCs were obtained by ESI-MS/MS analysis in positive ion mode, and 20 positional isomers of PCs were characterized in negative ion mode. Thus, this study presents a simple and powerful way to analyze fatty acyl residue composition and molecular species of PCs without destroying chemical structure.

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein.

Lipid Polarity Is Maintained in Absence of Tight Junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.

Axonal Gradient of Arachidonic Acid-containing Phosphatidylcholine and Its Dependence on Actin Dynamics

Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.

Erythrocyte lipid composition at different stages of type 1 diabetes in children

Complete profiles of phospholipid and ceramide molecular species from erythrocyte lipid extracts of children without carbohydrate metabolism disorders, and children with type 1 diabetes were compared by means of high performance liquid chromatography/mass spectrometry. For the first time a statistically significant increase (p<0.05) of lysophosphatidylcholine content in two groups of diabetic children with different duration of the disease (less than one year and more than one year) was found. Statistically significant changes in other lipid classes were not observed. The dependence of the content of phosphatidylcholine and phosphatidylethanolamine molecular species containing arachidonic acid residue (20:4) on the duration of the disease was found. The observed shift in lipid metabolism suggests of phospholipase A2 and chronic inflammatory process at different stages of diabetes mellitus, in cells (erythrocytes), which aer not involved in the immune response.

Imaging mass spectrometry reveals changes of metabolites distribution in mouse testis during testicular maturation

Although lipid metabolism is thought to be important for proper testicular maturation, the molecular mechanisms underlying this dynamic process remain incompletely understood. Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI‐IMS) can generate molecular ion images of tissue surfaces with high sensitivities and minimal chemical damage. With a wide range of detectable targets, we visualized various kinds of metabolites, such as glutathione, UDP‐HexNAc, phosphatidylcholine, sphingomyelin, phosphatidylinositol, seminolipid, and sulfatide. Here, we demonstrate the use of MALDI‐IMS with frozen sections of mouse testis at different developmental stages and clearly reveal that some molecular species of phosphatidylcholines and seminolipids show stage‐specific expression in testis. The result suggested that these molecular species serve different cellular functions during maturation. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Quantitative dissection of lipid degradation in rice seeds during accelerated aging

Aging during storage negatively affects rice seed viability and nutrition quality. Lipid degradation caused by phospholipase D (PLD) activity is known to be responsible for seed deterioration in Arabidopsis, but the mechanisms of this process in monocotyledonous plant rice remain unclear. In this study, we carried out lipid profiling analysis for rice seeds, and found that the main membrane lipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined during accelerated aging. In contrast, phosphatidic acid (PA), a hydrolysis product produced by PLD, increased. Interestingly, the degree of PA increase induced by accelerated aging (at 2 days) was much higher in rice than that in Arabidopsis (200% vs. 30%). Moreover, the decreased molecular species in PC and PE were well fit with those that were increased in PA, suggesting that PA may be derived from PLD-mediated lipid hydrolysis. OsPLDα1 and α3 were the major PLDαs in rice seeds, and their transcription increased significantly during the aging process. The aging treatment induced OsPLDα protein expression and activation. Three lipoxygenases (LOXs) were analyzed, and OsLOX2 transcription was induced significantly during seed aging. Our results reveal detailed information regarding lipid degradation during rice seed aging, and we go on to discuss the relationship between lipid degradation and peroxidation during seed aging.

Very-long-chain polyunsaturated fatty acids in the retina: analysis and clinical relevance in physiological and pathological conditions

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal glycerophospholipids, 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 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 (STGD3), a dominantly inherited juvenile macular degeneration leading to vision loss. In this context, PC molecular species containing VLC-PUFA in retina must be precisely characterized to improve our understanding of the pathogenesis of STGD3. For 25 years, several current approaches using gas chromatography or gas chromatography-mass spectrometry have been used for the characterisation and quantification of VLC-PUFA. These methods allowed characterizing with precision the structures (carbon chain length and double bond positions) of the different VLC-PUFA in bovine retina. Thus, retinal C28-C36 fatty acids are polyunsaturated and belong to the n-3 and n-6 families. The n-6 family of C28-C36 fatty acids has four or five double bonds, while the n-3 family of VLC-PUFA contains five or six double bonds. Moreover, some studies allowed determining the position of VLC-PUFA in the VLC-PC species. But most of these conventional approaches are time-consuming, requiring successive extraction, chromatographic steps and often a derivatization step before. Recently, a normal LC-ESI-MS/MS method was proposed for the structural characterization and the quantification of VLC-PC species in retinas from bovines and human donors. It directly analyses phospholipids as intact molecules and preserves the information based on the relative position of acyl radicals on the glycerol backbone. Since the results from molecular and biochemical studies led to the conclusion that VLC-PUFA are involved in the pathogenesis of STGD3, this specific and accurate method may be useful to more precisely investigate the molecular mechanisms leading to photoreceptor dysfunction and death in STD3.

Rapid analysis of surfactant phosphatidylcholine synthesis in preterm infants utilising the incorporation of a novel stable isotope: a pilot study

While introduction of exogenous surfactant therapy has dramatically improved survival of infants born 23–28 weeks gestation, the turnover of exogenous and synthesis of endogenous surfactant components are poorly understood in...

Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes

Stable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[ 2H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D −/− mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 μmol/lung/h) and SP-D −/− (0.69 μmol/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmol/lung/h, respectively). Increased lung PC in the SP-D −/− mouse was due to impaired catabolism, with a rate of accumulation of 0.057 μmol/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D −/− mice before significant PC acyl remodelling occurs.

Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry

Schizophrenia is one of the major psychiatric disorders, and lipids have focused on the important roles in this disorder. In fact, lipids related to various functions in the brain. Previous studies have indicated that phospholipids, particularly ones containing polyunsaturated fatty acyl residues, are deficient in postmortem brains from patients with schizophrenia. However, due to the difficulties in handling human postmortem brains, particularly the large size and complex structures of the human brain, there is little agreement regarding the qualitative and quantitative abnormalities of phospholipids in brains from patients with schizophrenia, particularly if corresponding brain regions are not used. In this study, to overcome these problems, we employed matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS), enabling direct microregion analysis of phospholipids in the postmortem brain of a patient with schizophrenia via brain sections prepared on glass slides. With integration of traditional histochemical examination, we could analyze regions of interest in the brain at the micrometric level. We found abnormal phospholipid distributions within internal brain structures, namely, the frontal cortex and occipital cortex. IMS revealed abnormal distributions of phosphatidylcholine molecular species particularly in the cortical layer of frontal cortex region. In addition, the combined use of liquid chromatography/electrospray ionization tandem mass spectrometry strengthened the capability for identification of numerous lipid molecular species. Our results are expected to further elucidate various metabolic processes in the neural system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-4909-3) contains supplementary material, which is available to authorized users.

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

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

Changes of Phospholipids in Duck Muscle by Different Heating Methods

We studied the changes in intramuscular phospholipids on boiling and roasting duck meat. Intramuscular lipids were distilled using chloroform and methanol. The phospholipids were separated using solid phase extraction (SPE) and analyzed with high performance liquid chromatography(HPLC). The fatty acid composition of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was determined using gas chromatography-mass spectroscopy(GC-MS), and the molecular species were analyzed using HPLC-MS. The results showed that the phospholipid content decreased on cooking, and the decrease was greater with roasting. The decrement in PE was greater than that in PC, primarily because the former contains more polyunsaturated fatty acids. The relative polyunsaturated fatty acid content and the PE and PC molecular species with polyunsaturated fatty acids all decreased.

Functional analysis of two isoforms of phosphatidylethanolamine N-methyltransferase

The enzyme catalysing the conversion of PE (phosphatidylethanolamine) into PC (phosphatidylcholine), PEMT (PE N-methyltransferase), exists as two isoforms, PEMT-L (longer isoform of PEMT) and PEMT-S (shorter isoform of PEMT). In the present study, to compare the functions of the two isoforms of PEMT, we established HEK (human embryonic kidney)-293 cell lines stably expressing PEMT-L and PEMT-S. Both PEMT-L and PEMT-S were localized in the ER (endoplasmic reticulum). PEMT-L, but not PEMT-S, was N-glycosylated with high-mannose oligosaccharides. The enzymatic activity of PEMT-S was much higher than that of PEMT-L. By using novel enzymatic assays for measuring PC and PE, we showed that PEMT-L and PEMT-S expression remarkably increased the cellular PC content, whereas the PE content was decreased by PEMT-S expression, but was hardly affected by PEMT-L expression. The cellular content of phosphatidylserine was also reduced by the expression of PEMT-L or PEMT-S. MS analyses demonstrated that the expression of PEMT-S led to more increases in the molecular species of PC and PC-O (ether-linked PC) with longer polyunsaturated chains than that of PEMT-L, whereas the PC-O species with shorter chains were increased more by PEMT-L expression than by PEMT-S expression, suggesting a difference in the substrate specificity of PEMT-L and PEMT-S. On the other hand, various PE and PE-O species were decreased by PEMT-S expression. In addition, PEMT-L and PEMT-S expression promoted the proliferation of HEK-293 cells. Based upon these findings, we propose a model in which the enzymatic activity and substrate specificity are regulated by the glycosylated N-terminal region of PEMT-L localized in the ER lumen.