Glycerophospholipid Profile Research Articles

Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington's Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HD gene, which encodes the protein Huntingtin. Huntingtin associates with membranes and can interact directly with glycerophospholipids in membranes. We analyzed glycerophospholipid profiles from brains of 11 month old wild-type (WT) and Q140/Q140 HD knock-in mice to assess potential changes in glycerophospholipid metabolism. Polar lipids from cerebellum, cortex, and striatum were extracted and analyzed by liquid chromatography and negative ion electrospray tandem mass spectrometry analysis (LC-MS/MS). Gene products involved in polar lipid metabolism were studied using western blotting, immuno-electron microscopy and qPCR. Significant changes in numerous species of glycerophosphate (phosphatidic acid, PA) were found in striatum, cerebellum and cortex from Q140/Q140 HD mice compared to WT mice at 11 months. Changes in specific species could also be detected for other glycerophospholipids. Increases in species of lyso-PA (LPA) were measured in striatum of Q140/Q140 HD mice compared to WT. Protein levels for c-terminal binding protein 1 (CtBP1), a regulator of PA biosynthesis, were reduced in striatal synaptosomes from HD mice compared to wild-type at 6 and 12 months. Immunoreactivity for CtBP1 was detected on membranes of synaptic vesicles in striatal axon terminals in the globus pallidus. These novel results identify a potential site of molecular pathology caused by mutant Huntingtin that may impart early changes in HD.

Glycerophospholipid Profiles of Bats with White-Nose Syndrome.

Pseudogymnoascus destructans is an ascomycetous fungus responsible for the disease dubbed white-nose syndrome (WNS) and massive mortalities of cave-dwelling bats. The fungus infects bat epidermal tissue, causing damage to integumentary cells and pilosebaceous units. Differences in epidermal lipid composition caused by P. destructans infection could have drastic consequences for a variety of physiological functions, including innate immune efficiency and water retention. While bat surface lipid and stratum corneum lipid composition have been described, the differences in epidermal lipid content between healthy tissue and P. destructans-infected tissue have not been documented. In this study, we analyzed the effect of wing damage from P. destructans infection on the epidermal polar lipid composition (glycerophospholipids [GPs] and sphingomyelin) of little brown bats (Myotis lucifugus). We hypothesized that infection would lead to lower levels of total lipid or higher oxidized lipid product proportions. Polar lipids from three damaged and three healthy wing samples were profiled by electrospray ionization tandem mass spectrometry. We found lower total broad lipid levels in damaged tissue, specifically ether-linked phospholipids, lysophospholipids, phosphatidylcholine, and phosphatidylethanolamine. Thirteen individual GP species from four broad GP classes were present in higher amounts in healthy tissue. Six unsaturated GP species were absent in damaged tissue. Our results confirm that P. destructans infection leads to altered lipid profiles. Clinical signs of WNS may include lower lipid levels and lower proportions of unsaturated lipids due to cellular and glandular damage.

A rapid LC–MS/MS method for quantitative profiling of fatty acids, sterols, glycerolipids, glycerophospholipids and sphingolipids in grapes

The abundance of lipids in plants is influenced by genotype and phenotype. Despite being a very important class of plant metabolites, knowledge of grape lipids is still very limited to date, with the exception of those located in seeds. Few investigations of grape lipids have shown that their profile depends on grape maturity, the variety and their location in the berry. Recent advances in liquid chromatography coupled to mass spectrometry have paved the way for faster analysis of lipids with minimal sample preparation. Here we describe a validation method for the extraction, identification and quantification of different classes of grape lipids: fatty acids, sterols, glycerolipids, glycerophospholipids and sphingolipids using liquid chromatographic electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The method was validated for 33 lipids, with linearity range (R2=0.95–1.00), LOQ (0.003–14.88ngmL−1) and intraday and interday repeatability being evaluated for each lipid. The lipid profiling method developed was successfully applied to the analysis of 18 grape samples (10 red grape and 8 white grape varieties) from 4 different genetic groups: Vitis vinifera, Vitis non-vinifera, Muscat and hybrid; 33 lipids were identified and quantified. This method, which can be easily expanded to include further compounds and other plant tissues, is the starting point for analysis of the lipid profile in different grape tissues, an essential goal for better understanding the role of lipids in grape physiology.

Aging-related Changes in Mouse Serum Glycerophospholipid Profiles

ObjectivesMetabolic dysfunction is a common hallmark of the aging process and aging-related pathogenesis. Blood metabolites have been used as biomarkers for many diseases, including cancers, complex chronic diseases, and neurodegenerative diseases. MethodsIn order to identify aging-related biomarkers from blood metabolites, we investigated the specific metabolite profiles of mouse sera from 4-month-old and 21-month-old mice by using a combined flow injection analysis–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry. ResultsAmong the 156 metabolites detected, serum levels of nine individual metabolites were found to vary with aging. Specifically, lysophosphatidylcholine (LPC) acyl (a) C24:0 levels in aged mice were decreased compared to that in young mice, whereas phosphatidylcholine (PC) acyl-alkyl (ae) C38:4, PC ae C40:4, and PC ae C42:1 levels were increased. Three classes of metabolites (amino acids, LPCs, and PCs) differed in intraclass correlation patterns of the individual metabolites between sera from young and aged mice. Additionally, the ratio of LPC a C24:0 to PC ae C38:4 was decreased in the aged mice, whereas the ratio of PC ae C40:4 to LPC a C24:0 was increased, supporting the aging-related metabolic changes of glycerophospholipids. ConclusionThe ratios of the individual metabolites PC and LPC could serve as potential biomarkers for aging and aging-related diseases.

Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or 125I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.

Progress in gene expression profiling by the introduction of metagenes

Identification of toxic mechanisms by test compoundinduced gene expression alterations is one of the major topics in current toxicological research (Ellinger-Ziegelbauer et al. 2008; Yamauchi et al. 2011; Bolt et al. 2010; Shimada et al. 2010; Adler et al. 2011; Heise et al. 2012). Also, a great deal of effort has been invested to optimize cell systems for gene array analyses in vitro (Godoy et al. 2009, 2010a, b; Zellmer et al. 2010; Hoehme et al. 2010). Recently, Schmidt and colleagues have introduced a technique that substantially facilitated the understanding and biological interpretation of complex gene expression profiles (Schmidt et al. 2008, 2009a, b, 2011, 2012): the metagenes of biological motifs. The authors observed sets of highly correlated genes in breast cancer tissue. Interestingly, these sets belonged to specific biological motifs, for example the proliferation metagene or a metagene of genes associated with the oestrogen receptor. Of particular interest are the newly discovered Band T-cell metagenes that indicate an infiltration of the tissue with immune cells. However, the presence of normal tissue can also be identified by a ‘normal-like metagene’. Further studies have reported that the mechanoactivity of tumour cells (Martin et al. 2012), stem cell properties (Lee et al. 2010), glycerophospholipid profiles (Cadenas et al. 2010) and apoptosis sensitivity (Petry et al. 2010) can be reflected by specific profiles of gene expression. Metagenes consist of sets of 30–100 genes and are usually calculated as the normalized mean of these genes. However, the analysis of such relatively high numbers of genes is laborious and expensive, a fact that becomes particularly relevant when the analysis is not performed by gene array but instead using qRT-PCR. The analysis of such high numbers of genes may not be necessary, as the genes within a metagene are highly correlated and contain redundant information. A recent study that aimed to reduce this complexity showed that analysis of a single gene (IGKC) can be as powerful in diagnosing B-cell infiltration as a metagene contains 60 individual genes (Schmidt et al. 2012). Another recently published study also contributed to metagene analysis by developing tools for statistical analysis of pre-clustered gene sets (Kammers et al. 2011). In addition, the selection of genes with a wide dynamic range or genes with particularly high expression differences has also been described (Hellwig et al. 2010). This offers the advantage that even high analytical variability does not compromise the interpretation of data. Although these developments have been made mostly in the field of cancer research, they may also be helpful for gene expression studies in toxicological research.

Glycerophospholipid profile in oncogene-induced senescence

Alterations in lipid metabolism and in the lipid composition of cellular membranes are linked to the pathology of numerous diseases including cancer. However, the influence of oncogene expression on cellular lipid profile is currently unknown. In this work we analyzed changes in lipid profiles that are induced in the course of ERBB2-expression mediated premature senescence. As a model system we used MCF-7 breast cancer cells with doxycycline-inducible expression of NeuT, an oncogenic ERBB2 variant. Affymetrix gene array data showed NeuT-induced alterations in the transcription of many enzymes involved in lipid metabolism, several of which (ACSL3, CHPT1, PLD1, LIPG, MGLL, LDL and NPC1) could be confirmed by quantitative realtime PCR. A study of the glycerophospholipid and lyso-glycerophospholipid profiles, obtained by high performance liquid chromatography coupled to Fourier-transform ion cyclotron resonance-mass spectrometry revealed senescence-associated changes in numerous lipid species, including mitochondrial lipids. The most prominent changes were found in PG(34:1), PG(36:1) (increased) and LPE(18:1), PG(40:7) and PI(36:1) (decreased). Statistical analysis revealed a general trend towards shortened phospholipid acyl chains in senescence and a significant trend to more saturated acyl chains in the class of phosphatidylglycerol. Additionally, the cellular cholesterol content was elevated and accumulated in vacuoles in senescent cells. These changes were accompanied by increased membrane fluidity. In mitochondria, loss of membrane potential along with altered intracellular distribution was observed. In conclusion, we present a comprehensive overview of altered cholesterol and glycerophospholipid patterns in senescence, showing that predominantly mitochondrial lipids are affected and lipid species less susceptible to peroxidation are increased.

Distinctive Glycerophospholipid Profiles of Human Seminoma and Adjacent Normal Tissues by Desorption Electrospray Ionization Imaging Mass Spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) has been successfully used to discriminate between normal and cancerous human tissue from different anatomical sites. On the basis of this, DESI-MS imaging was used to characterize human seminoma and adjacent normal tissue. Seminoma and adjacent normal paired human tissue sections (40 tissues) from 15 patients undergoing radical orchiectomy were flash frozen in liquid nitrogen and sectioned to 15μm thickness and thaw mounted to glass slides. The entire sample was two-dimensionally analyzed by the charged solvent spray to form a molecular image of the biological tissue. DESI-MS images were compared with formalin-fixed, hematoxylin and eosin (H&E) stained slides of the same material. Increased signal intensity was detected for two seminolipids [seminolipid (16:0/16:0) and seminolipid (30:0)] in the normal tubule testis tissue; these compounds were undetectable in seminoma tissue, as well as from the surrounding fat, muscle, and blood vessels. A glycerophosphoinositol [PI(18:0/20:4)] was also found at increased intensity in the normal testes tubule tissue when compared with seminoma tissue. Ascorbic acid (i.e., vitamin C) was found at increased amounts in seminoma tissue when compared with normal tissue. DESI-MS analysis was successfully used to visualize the location of several types of molecules across human seminoma and normal tissues. Discrimination between seminoma and adjacent normal testes tubules was achieved on the basis of the spatial distributions and varying intensities of particular lipid species as well as ascorbic acid. The increased presence of ascorbic acid within seminoma compared with normal seminiferous tubules was previously unknown.

Subcellular organelle lipidomics in TLR-4-activated macrophages

Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo(2)-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.

SLC1 and SLC4 Encode Partially Redundant Acyl-Coenzyme A 1-Acylglycerol-3-phosphate O-Acyltransferases of Budding Yeast

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.

Myocardial Lipidomics. Developments in myocardial nuclear lipidomics

The development of electrospray ionization mass spectrometry has been critical for the analyses of lipidomes from subcellular organelles. The myocardial nuclear lipidome likely has a key role in the molecular regulation of gene expression. In fact, recent studies have suggested that specific phospholipid classes bind and regulate specific transcription factors. The dynamic regulation of the myocardial nuclear lipidome may be critical in mediating long-term pathological responses to stresses such as ischemia, tachycardia, and hypertension. In this brief review, the preparation of myocardial nuclei is discussed, and the resulting nuclear lipidome from rat and rabbit are shown as examples. The rabbit myocardial nuclear lipidome contains relatively more plasmenylcholine/phosphatidylcholine molecular species in comparison to that ratio observed in the rat myocardial nuclear lipidome. The composition of the rat myocardial nuclear choline glycerophospholipid pool was relatively enriched with molecular species containing arachidonic acid and docosahexaenoic acid in comparison to that in the rabbit myocardial nuclear choline glycerophospholipid pool. While the ethanolamine glycerophospholipids of the rabbit myocardial nuclei are enriched with arachidonic acid and plasmalogens, the ethanolamine glycerophospholipid profile from rat myocardial nuclei show less plasmalogen and more species containing docosahexaenoic acid. Last, significant differences in the ethanolamine glycerophospholipid molecular species were observed in the rabbit heart lipidomes from the nucleus and the mitochondria. Quantitation of these lipid species in hearts subjected to pathophysiological stresses may provide important information on the role of the myocardial nuclear lipidome on long-term cardiac cell function.

Stearoyl-CoA Desaturase 1 Deficiency Increases CTP:Choline Cytidylyltransferase Translocation into the Membrane and Enhances Phosphatidylcholine Synthesis in Liver

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.

Changes in glycerophospholipid profile in experimental nephrotic syndrome

We investigated changes in the glycerophospholipids in kidney tissue and its various intracellular fractions in rats with nephrotic syndrome induced by puromycin aminonucleoside. The ethanolamine plasmalogen, 1- O-alk-1′-enyl-2-acyl-GPE (EP), was increased in kidney tissue obtained from the puromycin-treated animals. A similar increase was found in the mitochondria and endoplasmic reticulum (microsomes) of this tissue. These increases were not found in the liver. Since platelet-activating factor (PAF) is known to be produced in increased amounts in inflammatory disorders, it is suggested that the higher plasmalogen found in rat kidneys during experimental nephrotic syndrome might be derived from increased levels of this autacoid. The increase in PAF may also result in the elevation of plasma PAF-acetythydrolase (AH) activity observed in these animals.

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 · 10 −7 M), (3) with cortisol (1 · 10 −7 M) and (4) with thyroxine plus cortisol (1 · 10 −7 M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16 : 1( cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal Δ 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.

The phospholipid and fatty acid composition of human platelet surface and intracellular membranes isolated by high voltage free flow electrophoresis.

Human platelets were labeled with tracer doses of [14C]arachidonic acid, then fractionated into mixed membranes which are separated into intracellular membranes and two different domains of surface membranes by high voltage free flow electrophoresis. Each subfraction was analyzed for its phospholipid content. Glycerophospholipids were separated by high performance liquid chromatography and their fatty acids analyzed by glass capillary gas chromatography. Intracellular membranes appeared substantially depleted in sphingomyelin, while enrichment of this phospholipid was seen in surface membranes. PC and PI were more enriched in intracellular membranes than in the surface membranes and the contrary was observed for PE. On the other hand, the pattern of the phospholipid labeling by [14C]arachidonate followed closely the glycerophospholipid profiles of the membrane subfractions, but the specific radioactivity of PI was higher than of PC, which itself was higher than that of PE. Moreover, the endogenous content of arachidonic acid accentuates these tendencies. The percentage of arachidonate in PE was higher in the surface membranes than in the intracellular membranes and the contrary was observed for arachidonyl-PC and PI. These differences were compensated for by certain saturated and monounsaturated fatty acids present in the composition profiles. These findings are discussed in relation to the membrane localization of lipases involved in the liberation of arachidonic acid for prostanoid synthesis.