Phosphatidylethanolamine In Cells Research Articles

Biofilm formation and polar lipid biosynthesis in Mycobacterium abscessus are inhibited by naphthylmethylpiperazine.

Mycobacterium abscessus is a biofilm-forming, non-tuberculous mycobacterium that is highly resistant to antibiotics. Bacterial efflux pumps contribute to biofilm formation, export of biofilm-associated lipids and antibiotic tolerance. The Resistance Nodulation Cell Division (RND) and ATP-Binding Cassette (ABC) families of efflux pumps export lipids to the mycobacterial cell surface. 1-(1-naphthyl methyl)-piperazine (NMP) is a chemosensitizer that causes membrane destabilization and is an inhibitor of RND efflux pumps. The effects of NMP on biofilm formation and lipid metabolism in M. abscessus biofilms have not been investigated. Plumbagin (PLU) is an inhibitor of ABC efflux pumps that has not been studied for its effects on antibiotic tolerance in M. abscessus biofilms. In this study, we report that the efflux pump inhibitors NMP and PLU inhibit biofilm formation by 50% at sub-MIC levels. We show that NMP inhibits the incorporation of the radiolabeled long-chain fatty acid 14C-palmitate into glycopeptidolipids in cell surface lipids of log-phase M. abscessus. NMP also inhibits the utilization of the radiolabel in the biosynthesis of phosphatidylethanolamine in the cell surface and cellular lipids of M. abscessus cells in log-phase and in biofilms. Incorporation of the radiolabel into cardiolipin in the cellular lipids of M. abscessus biofilms was inhibited by NMP. The incorporation of 14C-acetate into cell surface phosphatidylethanolamine in log-phase and biofilm cells was also inhibited by NMP. Triacylglycerol biosynthesis using 14C-palmitate and 14C-acetate in cellular lipids of log-phase and biofilm cells was increased several folds by NMP. Efflux pump activity in M. abscessus cells was inhibited by 97% and 68% by NMP and PLU respectively. NMP and PLU caused 5-fold decreases in the minimum inhibitory concentrations of ciprofloxacin and clarithromycin against M. abscessus. Our results demonstrate that NMP and PLU affect important physiological processes in M. abscessus associated with its pathogenesis.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

SLC27A4-mediated selective uptake of mono-unsaturated fatty acids promotes ferroptosis defense in hepatocellular carcinoma.

Aberrant lipid metabolism mediated by the selective transport of fatty acids plays vital roles in cancer initiation, progression, and therapeutic failure. However, the biological function and clinical significance of abnormal fatty acid transporters in human cancer remain unclear. In the present study, we reported that solute carrier family 27 member 4 (SLC27A4) is significantly overexpressed in 21 types of human cancer, especially in the fatty acids-enriched microenvironment surrounding hepatocellular carcinoma (HCC), breast cancer, and ovarian cancer. Upregulated SLC27A4 expression correlated with shorter overall and relapse-free survival of patients with HCC, breast cancer, or ovarian cancer. Lipidomic analysis revealed that overexpression of SLC27A4 significantly promoted the selective uptake of mono-unsaturated fatty acids (MUFAs), which induced a high level of MUFA-containing phosphatidylcholine and phosphatidylethanolamine in HCC cells, consequently resulting in resistance to lipid peroxidation and ferroptosis. Importantly, silencing SLC27A4 significantly promoted the sensitivity of HCC to sorafenib treatment, both in vitro and in vivo. Our findings revealed a plausible role for SLC27A4 in ferroptosis defense via lipid remodeling, which might represent an attractive therapeutic target to increase the effectiveness of sorafenib treatment in HCC.

Specificity of ABCA7-mediated cell lipid efflux

Adenosine triphosphate-binding cassette transporter subfamily A member 7 (ABCA7) performs incompletely understood biochemical functions that affect pathogenesis of Alzheimer's disease. ABCA7 is most similar in primary structure to ABCA1, the protein that mediates cell lipid efflux and formation of high-density lipoprotein (HDL). Lipid metabolic labeling/tracer efflux assays were employed to investigate lipid efflux in BHK-ABCA7(low expression), BHK-ABCA7(high expression) and BHK-ABCA1 cells. Shotgun lipid mass spectrometry was used to determine lipid composition of HDL synthesized by BHK-ABCA7 and BHK-ABCA1 cells. BHK-ABCA7(low) cells exhibited significant efflux only of choline-phospholipid and phosphatidylinositol. BHK-ABCA7(high) cells had significant cholesterol and choline-phospholipid efflux to apolipoprotein (apo) A-I, apo E, the 18A peptide, HDL, plasma and cerebrospinal fluid and significant efflux of sphingosine-lipid, serine-lipid (which is composed of phosphatidylserine and phosphatidylethanolamine in BHK cells) and phosphatidylinositol to apo A-I. In efflux assays to apo A-I, after adjustment to choline-phospholipid, ABCA7-mediated efflux removed ~4 times more serine-lipid and phosphatidylinositol than ABCA1-mediated efflux, while ABCA1-mediated efflux removed ~3 times more cholesterol than ABCA7-mediated efflux. Shotgun lipidomic analysis revealed that ABCA7-HDL had ~20 mol% less phosphatidylcholine and 3–5 times more serine-lipid and phosphatidylinositol than ABCA1-HDL, while ABCA1-HDL contained only ~6 mol% (or ~1.1 times) more cholesterol than ABCA7-HDL. The discrepancy between the tracer efflux assays and shotgun lipidomics with respect to cholesterol may be explained by an underestimate of ABCA7-mediated cholesterol efflux in the former approach. Overall, these results suggest that ABCA7 lacks specificity for phosphatidylcholine and releases significantly but not dramatically less cholesterol in comparison with ABCA1.

Plasma and red blood cell n3 fatty acids correlate positively with the WISC-R verbal and full-scale intelligence quotients and inversely with Conner's parent-rated ADHD index t-scores in children with high functioning autism and Asperger's syndrome

Findings of the fatty acid status of people with autism spectrum disorders have been incongruent perhaps because of the diversity of the condition. A cross-sectional design study was used to investigated fatty acid levels and relationships between fatty acids, and cognition and behaviour in a homogenous group of children with autism spectrum disorder. Children with Asperger's syndrome (AS) /high functioning autism (n = 44) and healthy siblings (n = 17) were recruited from the Diagnostic and Therapeutic Centre for Children with Autism, Warsaw, Poland. In the AS group, plasma phosphatidylcholine 22:5n3 correlated positively with verbal (r = 0.357, p = 0.019) and full scale (r = 0.402, p = 0.008) IQs, red blood cell phosphatidylcholine 22:5n3 with verbal (r = 0.308, p = 0.044), performance (r = 0.304, p = 0.047) and full scale (r = 0.388, p = 0.011) IQs and red blood cell phosphatidylethanolamine 22:5n3 with verbal (r = 0.390, p = 0.010) and full scale (r = 0.370, p = 0.016) IQs. Whilst, plasma phosphatidycholine 20:5n3 (r = -0.395, p = 0.009), 22:6n3 (r = -0.402, p = 0.007) and total n3 fatty acids (r = -425, p = 0.005), red blood cell phosphatidlycholine 20:5n3 (r = -0.321, p = 0.036) and red blood cell phosphatidylethanolamine 20:5n3 (r = -0.317, p = 0.038), 22:6n3 (r = -0.297, p = 0.05) and total n3 fatty acids (r = -0.306, p = 0.046) correlated inversly with ADHD index. Similarly, inattention was negatively related with plasma phosphatidylcholine 22:6n3 (r = -0.335, p = 0.028), and total n3 fatty acids (r = -0.340, p = 0.026), oppositional with plasma phosphatidylcholine 18:3n3 (r = -0.333, p = 0.029), 20:5n3 (r = -0.365, p = 0.016), total n3 fatty acids (r = -0.293, p < 0.05), red blood cell phosphatidylcholine 18:3n3 (r = -0.337, p = 0.027) and red blood cell ethanolamine 18:3n3 (r = - 0.333, p = 0.029), 20:5n3 (r = -0.328, p = 0.032), 22:6n3 (r = 0.362, p = 0.017) and total n-3 fatty acids (r = -0.298, p < 0.05) and hyperactivity with plasma phosphatidylcholine 22:6n3 (r = -0.320, p = 0.039). In contrast, there were inverse correlations between red blood cell phosphatidylcholine 18:2n6 and performance (r = -0.358, p = 0.019) and full scale (r = -0.320, p = 0.039) IQs, and direct correlations between red blood cell phosphatidylcholine 22:4n6 (r = 0.339, p = 0.026) and 22:5n6 (r = 0.298, p < 0.05) and ADHD index, between red blood cell phosphatidylcholine 22:4n6 (r = 0.308, p = 0.044) and inattention, between plasma phosphatidylcholine 22:4n6 (r = 0.341, p = 0.025), red blood cell phosphatidylcholine 20:4n6 (r = 0.314, p = 0.041) and total n6 fatty acids (r = 0.336, p = 0.028) and oppositional and plasma phosphatidylcholine 20:3n6 (r = 0.362, p = 0.018) and red blood cell phosphatidylcholine 20:3n6 (r = 0.401, p = 0.009) and hyperactivity. The findings of the ethnically homogenous children with Asperger's syndrome/high functioning autism study revealed positive associations between 22:5n3 and cognition, and negative relationships between 20:5n3 and 22:6n3 and behavioural problem. In contrast, cognitive ability and behavioural problems were negatively and positively associated with n6 fatty acids. Further investigation is required to establish whether there a cause and effect relationship. Regardless, it would be prudent to ensure that children with the conditions have optimum n3 PUFA intake.

Elevation of homocysteine levels in the plasma leads to deregulation of lipid metabolism in a rabbit model of atherosclerosis

Cardiovascular disease, the leading cause of death worldwide, is in the majority of cases a result of atherosclerosis, which can be only to 50% explained by established risk factors including hypercholesterolemia (HCL). Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. It is linked to numerous human diseases, strongly correlates with cardiovascular and all-cause mortality, and is associated with deregulation of lipid metabolism. HHcy is found in 5-10% of the general population and up to 30% of the elderly, and is mainly due to genetic defects or vitamin deficiency resulting in blocking of homocysteine (Hcy) degradation. We induced HHcy by dietary partial blocking of Hcy degradation and intravenous injections of Hcy into a rabbit model of atherosclerosis in the presence or absence of HCL. Deficiency of vitamins and choline required for Hcy degradation in the absence of HCL leads to an accumulation of lipid droplets in cells of the aorta including endothelial cells, smooth muscle cells, and fibroblasts, an accumulation of dilated ER in fibroblasts of the aorta as well as decreased LPC18:1 and 18:2 levels in muscles. In addition, while dietary blocking of Hcy degradation results in increased triacylglycerol (TAG) concentrations in blood cells, further elevation of Hcy levels leads to their decrease, but also to ceramide, lyso-phosphatidylcholine (LPC), phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin accumulation in blood cells, compared to the diet blocking Hcy degradation alone. A decrease of TAG levels was also observed in the liver, in response to intravenous injections of Hcy and in rabbits receiving a combined diet that blocked Hcy degradation and induced HCL. In conclusion our data suggest that dietary blocking of Hcy degradation as well as Hcy itself may contribute to different human pathologies by deregulation of lipid metabolism in different tissues.

A regulatory role of scavenger receptor class B type 1 in endocytosis and lipid droplet formation induced by liposomes containing phosphatidylethanolamine in HEK293T cells

We have recently reported that phosphatidylethanolamine (PE)-containing liposomes are endocytosed and then induce lipid droplets (LDs) in HEK293T cells. In this study, we elucidated a mechanism responsible for endocytosis of PE-containing liposomes and induction of LDs. By using fluorescence-labeled liposomes and flow cytometry, we found that PE-containing liposomes were very efficiently internalized in HEK293T cells. However, Block lipid transporter-1 (BLT-1) only marginally suppressed the uptake of these liposomes, indicating that entire liposomes were mostly taken up in these cells. They were therefore inferred to express abundant PE receptors responsible for endocytosis of PE-containing liposomes. We examined the expression of 52 candidate genes through transcriptomic analyses and eventually narrowed it down to four candidate genes, which were abundantly expressed in HEK293T cells. Among siRNAs targeting these candidates, scavenger receptor class B type 1 (SR-B1) siRNA showed the most profound reduction in PE liposomal uptake. Conversely, the expression of SR-B1 by transfection of an expression plasmid enhanced the uptake of PE-containing liposomes. After the internalization of PE-containing liposomes, they were colocalized with endosomes/lysosomes and SR-B1, which indicates that these liposomes are taken up in HEK293T cells at least partially through the endosomal/lysosomal pathway. A specific anti-SR-B1-antibody blocked the uptake of PE-containing liposomes in HEK293T cells while LD formation in these cells induced by PE-containing liposomes was suppressed by treatment with SR-B1 siRNA. These results demonstrate that SR-B1 functions as a receptor for the endocytosis of PE-containing liposomes and regulates the formation of LDs induced by PE-containing liposomes in HEK293T cells.

Dual-Isotope SPECT Imaging with NIS Reporter Gene and Duramycin to Visualize Tumor Susceptibility to Oncolytic Virus Infection

Noninvasive dual-imaging methods that provide an early readout on tumor permissiveness to virus infection and tumor cell death could be valuable in optimizing development of oncolytic virotherapies. Here, we have used the sodium iodide symporter (NIS) and 125I radiotracer to detect infection and replicative spread of an oncolytic vesicular stomatitis virus (VSV) in VSV-susceptible (MPC-11 tumor) versus VSV-resistant (CT26 tumor) tumors in BALB/c mice. In conjunction, tumor cell death was imaged simultaneously using technetium (99mTc)-duramycin that binds phosphatidylethanolamine in apoptotic and necrotic cells. Dual-isotope single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed areas of virus infection (NIS and 125I), which overlapped well with areas of tumor cell death (99mTc-duramycin imaging) in susceptible tumors. Multiple infectious foci arose early in MPC-11 tumors, which rapidly expanded throughout the tumor parenchyma over time. There was a dose-dependent increase in numbers of infectious centers and 99mTc-duramycin-positive areas with viral dose. In contrast, NIS or duramycin signals were minimal in VSV-resistant CT26 tumors. Combinatorial use of NIS and 99mTc-duramycin SPECT imaging for simultaneous monitoring of oncolytic virotherapy (OV) spread and the presence or absence of treatment-associated cell death could be useful to guide development of combination treatment strategies to enhance therapeutic outcome.

Metabolite profiles of callus and cell suspension cultures of mangosteen.

Callus was induced from mangosteen (Garcinia mangostana L.) young purple-red leaves on Murashige and Skoog basal medium with various combinations of plant growth regulators. Murashige and Skoog medium with 4.44µM 6-benzylaminopurine and 4.52µM 2,4-dichlorophenoxyacetic acid was the best for friable callus induction. This friable callus was used for the initiation of cell suspension culture. The effects of different combinations of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid, carbon sources and inoculum sizes were tested. It was found that combination of 2.22µM 6-benzylaminopurine + 2.26µM 2,4-dichlorophenoxyacetic acid, glucose (30g/l) and 1.5g/50ml inoculum size was the best for cell growth. Callus and cell suspension cultures were then treated either with 100µM methyl jasmonate as an elicitor for 5 days, or 0.5g/l casein hydrolysate as an organic supplement for 7 days. Metabolites were then extracted and profiled using liquid chromatography-time of flight mass spectrometry. Multivariate discriminant analyses revealed significant metabolite differences (P ≤ 0.05) for callus and suspension cells treated either with methyl jasmonate or casein hydrolysate. Based on MS/MS data, methyl jasmonate stimulated the production of an alkaloid (thalsimine) and fatty acid (phosphatidyl ethanolamine) in suspension cells while in callus, an alkaloid (thiacremonone) and glucosinolate (7-methylthioheptanaldoxime) was produced. Meanwhile casein hydrolysate stimulated the production of alkaloids such as 3ß,6ß-dihydroxynortropane and cis-hinokiresinol and triterpenoids such as schidigerasaponin and talinumoside in suspension cells. This study provides evidence on the potential of secondary metabolite production from in vitro culture of mangosteen.

Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Effect of docosahexaenoic acid-enriched fish oil supplementation in pregnant women with Type 2 diabetes on membrane fatty acids and fetal body composition--double-blinded randomized placebo-controlled trial.

To test if docosahexaenoic acid-enriched fish oil supplementation rectifies red cell membrane lipid anomaly in pregnant women with Type2 diabetes and their neonates, and alters fetal body composition. Women with Type2 diabetes (n=88; 41 fish oil, 47 placebo) and healthy women (n=85; 45 fish oil, 40 placebo) were supplemented from the first trimester until delivery. Blood fatty acid composition, fetal biometric and neonatal anthropometric measurements were assessed. A total of 117 women completed the trial. The women with Type2 diabetes who took fish oil compared with those who received placebo had higher percentage of docosahexaenoic acid in red cell phosphatidylethanolamine in the third trimester (12.0% vs. 8.9%, P=0.000) and at delivery (10.7% vs. 7.4%, P=0.001). Similarly, the neonates of the women with Type2 diabetes supplemented with the fish oil had increased docosahexaenoic acid in the red cell phosphatidylethanolamine (9.2% vs. 7.7%, P=0.027) and plasma phosphatidylcholine (6.1% vs. 4.7%, P=0.020). Docosahexaenoic acid-rich fish oil had no effect on the body composition of the fetus and neonates of the women with Type2 diabetes. A daily dose of 600mg of docosahexaenoic acid was effective in ameliorating red cell membrane docosahexaenoic acid anomaly in pregnant women with Type2 diabetes and neonates, and in preventing the decline of maternal docosahexaenoic acid during pregnancy. We suggest that the provision of docosahexaenoic acid supplement should be integrated in the antenatal care of pregnant women with Type2 diabetes.

Manipulation of phospholipid composition alters protein binding to lipid droplets (606.3)

Lipid droplets are cytoplasmic organelles that store excess lipid. Each lipid droplet consists of a core of neutral lipid surrounded by a phospholipid monolayer. Proteins interact with the phospholipid surface and regulate lipid droplet synthesis and degradation. We have shown that the phospholipid composition of synthetic lipid droplets can influence protein binding in an in vitro assay. We aimed to determine if protein binding to lipid droplets is influenced by the lipid droplet phospholipid composition in cells. We altered the phospholipid composition of lipid droplets in NIH3T3 cells with hexadecylphosphocholine (HePC), an inhibitor of phosphatidylcholine (PC) synthesis. Isolation of lipid droplets from inhibitor treated and control cells and separation of phospholipids by thin layer chromatography, demonstrated a decrease in PC relative to phosphatidylethanolamine in HePC‐treated cells. Furthermore, HePC treatment prevented the lipid droplet localization of CTP:phosphocholine cytidylyltransferase, the enzyme that catalyzes the rate‐limiting step in PC synthesis. Other changes in protein localization to lipid droplets were observed by SDS‐PAGE and silver staining of lipid droplet proteins in HePC‐treated and control cells. Current and future experiments continue to examine how changes in phospholipid composition alter the protein composition of this important storage compartment.Grant Funding Source: Supported by an award from Research Corporation for Science Advancement

Dietary fat intake, circulating and membrane fatty acid composition of healthy Norwegian men and women

The present study aimed to assess the dietary fat intake and blood fatty acid status of healthy Norwegian men and women living in Bergen whose habitual diet is known to be high in long-chain omega-3 fat. Healthy men (n=41) and women (n=40) aged 20-50years who were regular blood donors completed 7-day food diaries and their nutrient intake was analysed by Norwegian food database software, kbs, version 4.9 (kostberegningssystem; University of Oslo, Oslo, Norway). Blood samples were obtained before blood donation and assessed for the fatty acid composition of plasma triglycerides and cholesterol esters, phosphatidylcholine, and red cell phosphatidylcholine and phosphatidylethanolamine. There was no difference in dietary fat intake between men and women. Total and saturated fat intakes exceeded the upper limits of the recommendations of the National Nutrition Council of Norway. Although polyunsaturated fat intake was close to the lower limit of the recommended level, the intake varied greatly among individuals, partly as a result of the use of supplementary fish oil. Moreover, the proportional fatty acid composition of plasma and red cell lipids was similar between men and women. Enrichment of docosahexaenoic acid in red cell phosphatidylethanolamine was found in fish oil users. The results of the present study provide a snapshot of the current nutritional status of healthy Norwegian adults. Moreover, the detailed blood fatty acid composition of men and women whose habitual diet constitutes high long-chain polyunsaturated omega-3 fat as well as saturated fat could be used as reference value for population studies.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue

In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H(2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E(2) and LGD(2). These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD(2) (ox-LGD(2)) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD(2) was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD(2) is a common oxidized metabolite of LGD(2). In the planar structure of ox-LGD(2), H8 and H12 of LGD(2) were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD(2) is less reactive with amines than LGs. Therefore, ox-LGD(2) might be considered a detoxification metabolite of LGD(2).

Differential effect of maternal diet supplementation with α-Linolenic adcid or n-3 long-chain polyunsaturated fatty acids on glial cell phosphatidylethanolamine and phosphatidylserine fatty acid profile in neonate rat brains

BackgroundDietary long-chain polyunsaturated fatty acids (LC-PUFA) are of crucial importance for the development of neural tissues. The aim of this study was to evaluate the impact of a dietary supplementation in n-3 fatty acids in female rats during gestation and lactation on fatty acid pattern in brain glial cells phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the neonates.MethodsSprague-Dawley rats were fed during the whole gestation and lactation period with a diet containing either docosahexaenoic acid (DHA, 0.55%) and eicosapentaenoic acid (EPA, 0.75% of total fatty acids) or α-linolenic acid (ALA, 2.90%). At two weeks of age, gastric content and brain glial cell PE and PS of rat neonates were analyzed for their fatty acid and dimethylacetal (DMA) profile. Data were analyzed by bivariate and multivariate statistics.ResultsIn the neonates from the group fed with n-3 LC-PUFA, the DHA level in gastric content (+65%, P < 0.0001) and brain glial cell PE (+18%, P = 0.0001) and PS (+15%, P = 0.0009) were significantly increased compared to the ALA group. The filtered correlation analysis (P < 0.05) underlined that levels of dihomo-γ-linolenic acid (DGLA), DHA and n-3 docosapentaenoic acid (DPA) were negatively correlated with arachidonic acid (ARA) and n-6 DPA in PE of brain glial cells. No significant correlation between n-3 and n-6 LC-PUFA were found in the PS dataset. DMA level in PE was negatively correlated with n-6 DPA. DMA were found to occur in brain glial cell PS fraction; in this class DMA level was correlated negatively with DHA and positively with ARA.ConclusionThe present study confirms that early supplementation of maternal diet with n-3 fatty acids supplied as LC-PUFA is more efficient in increasing n-3 in brain glial cell PE and PS in the neonate than ALA. Negative correlation between n-6 DPA, a conventional marker of DHA deficiency, and DMA in PE suggests n-6 DPA that potentially be considered as a marker of tissue ethanolamine plasmalogen status. The combination of multivariate and bivariate statistics allowed to underline that the accretion pattern of n-3 LC-PUFA in PE and PS differ.

Thematic Review Series: Glycerolipids. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are two aminophospholipids whose metabolism is interrelated. Both phospholipids are components of mammalian cell membranes and play important roles in biological processes such as apoptosis and cell signaling. PS is synthesized in mammalian cells by base-exchange reactions in which polar head groups of preexisting phospholipids are replaced by serine. PS synthase activity resides primarily on mitochondria-associated membranes and is encoded by two distinct genes. Studies in mice in which each gene has been individually disrupted are beginning to elucidate the importance of these two synthases for biological functions in intact animals. PE is made in mammalian cells by two completely independent major pathways. In one pathway, PS is converted into PE by the mitochondrial enzyme PS decarboxylase. In addition, PE is made via the CDP-ethanolamine pathway, in which the final reaction occurs on the endoplasmic reticulum and nuclear envelope. The relative importance of these two pathways of PE synthesis has been investigated in knockout mice. Elimination of either pathway is embryonically lethal, despite the normal activity of the other pathway. PE can also be generated from a base-exchange reaction and by the acylation of lyso-PE. Cellular levels of PS and PE are tightly regulated by the implementation of multiple compensatory mechanisms.

Increased levels of mercury associated with high fish intakes among children from Vancouver, Canada

To assess exposure to mercury (Hg) among children in population subgroups whose traditional dietary practices include fish. We determined blood Hg, red blood cell phosphatidylethanolamine omega-3 eicosapentaenoic acid as a marker of fish intake, and assessed indexes of childhood behavior in preschool children 1.5 to 5 years of age (n = 228) living in an ethnically diverse neighborhood in Vancouver, British Columbia, Canada. The median blood Hg was 4.6 nmol/L, range 0-67.9 nmol/L. Twelve (6%) children, all of whom were Chinese, had a blood Hg > 28.9 nmol/L. Blood Hg, total fish intake, and eicosapentaenoic acid were higher among Chinese than Caucasian children; however, higher fish intake did not predict blood Hg. Blood Hg was inversely associated with attentional focusing in children over 3 years of age after adjusting for confounding family variables, iron deficiency anemia, and zinc deficiency. Major sources of fish among Chinese children were imported fish rather than local fish. Children from population subgroups within populations not considered at risk may be at increased risk of neurotoxicity caused by Hg exposure from fish.

Attenuated secretion of very low density lipoproteins from McA-RH7777 cells treated with eicosapentaenoic acid is associated with impaired utilization of triacylglycerol synthesized via phospholipid remodeling

In McA-RH7777 cells stably expressing human apolipoprotein (apo) B100, treatment with oleic acid (18:1(n-9)) promoted whereas treatment with eicosapentaenoic acid (EPA, 20:5(n-3)) attenuated assembly and secretion of VLDL. Under conditions where the cells were cultured in the presence of 20% serum, EPA (0.4 mM) had marginal effect on the secretion of total apoB100 (determined by pulse-chase analysis) but decreased (by 50%) secretion of triacylglycerol (TG), indicating that the inhibitory effect of EPA was exerted primarily on TG-rich VLDL. Analysis of phospholipid mass and species by tandem mass spectrometry showed increased phosphatidylethanolamine (PE) in EPA-treated cells, the increase was significant in the distal Golgi membranes (by 170%) and endoplasmic reticulum (by 116%). Lipid pulse-chase studies showed a major distinction between phospholipid species containing 20:5(n-3) and 18:1(n-9), which in turn was associated with distinct compartmentalization of TG containing 20:5(n-3) or 18:1(n-9) between cytosol and microsomes and their recruitment during VLDL assembly. Thus, 18:1-TG was secreted as VLDL but 20:5-TG was not. These results suggest that EPA attenuation of VLDL secretion is associated with impaired utilization of TG derived from phospholipid remodeling.

Disruption of the Phosphatidylserine Decarboxylase Gene in Mice Causes Embryonic Lethality and Mitochondrial Defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.