Phosphatidylcholine Species Research Articles

Postprandial remodeling of high-density lipoprotein following high saturated fat and high carbohydrate meals

Humans spend most of the time in the postprandial state, yet most knowledge about high-density lipoproteins (HDL) derives from the fasted state. HDL protein and lipid cargo mediate HDL's antiatherogenic effects, but whether these HDL constituents change in the postprandial state and are affected by dietary macronutrients remains unknown. This study aimed to assess changes in HDL protein and lipid composition after the consumption of a high-carbohydrate or high saturated fat (HSF) meal. We isolated HDL from plasma collected during a randomized, cross-over study of metabolically healthy subjects. Subjects consumed isocaloric meals consisting predominantly of either carbohydrate or fat. At baseline and at 3 and 6hours postprandial, we quantified HDL protein and lipid composition by liquid chromatography-mass spectrometry. A total of 15 subjects were included (60% female, aged 34±15years, body mass index: 24.1±2.7kg/m2). Consumption of the HSF meal led to HDL enrichment in total lipid (P=.006), triglyceride (P=.02), and phospholipid (P=.008) content and a corresponding depletion in protein content. After the HSF meal, 16 of the 25 measured phosphatidylcholine species significantly increased in abundance (P values range from .027 to <.001), along with several sphingolipids including ceramides (P<.004), lactosylceramide (P=.023), and sphingomyelin-14 (P=.013). Enrichment in apolipoprotein A-I (P=.001) was the only significant change in HDL protein composition after the HSF meal. The high-carbohydrate meal conferred only minimal changes in HDL composition. Meal macronutrient content acutely affects HDL composition in the postprandial state, with the HSF meal resulting in enrichment of HDL phospholipid content with possible consequences for HDL function.

Lipidomic analysis reveals sphingomyelin and phosphatidylcholine species associated with renal impairment and all-cause mortality in type 1 diabetes

There is an urgent need for a better molecular understanding of the pathophysiology underlying development and progression of diabetic nephropathy. The aim of the current study was to identify novel associations between serum lipidomics and diabetic nephropathy. Non-targeted serum lipidomic analyses were performed with mass spectrometry in 669 individuals with type 1 diabetes. Cross-sectional associations of lipid species with estimated glomerular filtration rate (eGFR) and urinary albumin excretion were assessed. Moreover, associations with register-based longitudinal follow-up for progression to a combined renal endpoint including ≥30% decline in eGFR, ESRD and all-cause mortality were evaluated. Median follow-up time was 5.0–6.4 years. Adjustments included traditional risk factors and multiple testing correction. In total, 106 lipid species were identified. Primarily, alkyl-acyl phosphatidylcholines, triglycerides and sphingomyelins demonstrated cross-sectional associations with eGFR and macroalbuminuria. In longitudinal analyses, thirteen lipid species were associated with the slope of eGFR or albuminuria. Of these lipids, phosphatidylcholine and sphingomyelin species, PC(O-34:2), PC(O-34:3), SM(d18:1/24:0), SM(d40:1) and SM(d41:1), were associated with lower risk of the combined renal endpoint. PC(O-34:3), SM(d40:1) and SM(d41:1) were associated with lower risk of all-cause mortality while an SM(d18:1/24:0) was associated with lower risk of albuminuria group progression. We report distinct associations between lipid species and risk of renal outcomes in type 1 diabetes, independent of traditional markers of kidney function.

Distribution of plasma oxidised phosphatidylcholines in chronic kidney disease and periodontitis as a co-morbidity

Individuals with chronic kidney disease (CKD) and periodontitis as a co-morbidity have a higher mortality rate than individuals with CKD and no periodontitis. The inflammatory burden associated with both diseases contributes to an increased risk of cardiovascular and all-cause mortality. We previously demonstrated that periodontitis is associated with increasing circulating markers of inflammation and oxidative stress. We propose that inflammatory oxidised phosphocholines may contribute to the increased risk of cardiovascular disease in patients with CKD. However, the analysis of oxidised phospholipids has been limited by a lack of authentic standards for absolute quantification. Here, we have developed a comprehensive quantification liquid chromatography-mass spectrometry-based multiple reaction monitoring method for oxidised phospholipids (including some without available authentic species) that enables us to simultaneously measure twelve oxidised phosphatidylcholine species with high levels of sensitivity and specificity. The standard curves for commercial standards 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylcholine (PGPC); 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphatidylcholine (PONPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphatidylcholine (PAzPC) and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphatidylcholine (POVPC), were linear with a correlation coefficient greater than 0.99 for all analytes. The method is reproducible, with intra- and inter-day precision <15%, and accuracy within ±5% of nominal values for all analytes. This method has been successfully applied to investigate oxidised phosphatidylcholine in plasma from CKD patients with and without chronic periodontitis and the data that was obtained has been compared to plasma from healthy controls. Comparative analysis demonstrates altered chain fragmented phosphatidylcholine profiles in the plasma samples of patients with CKD and periodontitis as a co-morbidity compared to healthy controls.

Impact of Frying on Changes in Clam (Ruditapes philippinarum) Lipids and Frying Oils: Compositional Changes and Oxidative Deterioration

AbstractThe current study shows the compositional changes and oxidation development of clam (Ruditapes philippinarum, R. philippinarum) lipids and frying oils when subjected to different processing conditions. Parameters measured include acid value, peroxide value (POV), thiobarbituric acid‐reactive substances (TBARS), total oxidation (TOTOX), lipid classes, fatty acid composition, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) contents together with major glycerophospholipid (GP) molecular species. Deep‐fat frying increased triacylglycerol (TAG) content and decreased the contents of PC, PE, and GP molecular species in clam in a time‐dependent manner. Meanwhile, minor amounts of free fatty acids, diacylglycerols, monoacylglycerols, and polar lipids were detected in frying oils, indicating lipid migration between the clam and frying oils. The time‐dependent increase of POV, TBARS, and TOTOX in fried clams and frying oils with concurrent reduction of docosahexenoic acid and eicosapentaenoic acid indicates extensive oxidative degradation of clam lipids. Moreover, the moisture‐rich clam aggravated the deterioration of frying oils. Consequently, deep‐fat frying significantly altered the lipid profile and decreased the nutritional value of clams.

Variations in hepatic lipid species of age-matched male mice fed a methionine-choline-deficient diet and housed in different animal facilities

BackgroundNon-alcoholic steatohepatitis (NASH) is a common disease and feeding mice a methionine-choline-deficient (MCD) diet is a frequently used model to study its pathophysiology. Genetic and environmental factors influence NASH development and liver lipid content, which was studied herein using C57BL/6 J mice bred in two different animal facilities.MethodsAge-matched male C57BL/6 J mice bred in two different animal facilities (later on referred to as WT1 and WT2) at the University Hospital of Regensburg were fed identical MCD or control chows for 2 weeks. Hepatic gene and protein expression and lipid composition were determined.ResultsNASH was associated with increased hepatic triglycerides, which were actually higher in WT1 than WT2 liver in both dietary groups. Cholesterol contributes to hepatic injury but was only elevated in WT2 NASH liver. Ceramides account for insulin resistance and cell death, and ceramide species d18:1/16:0 and d18:1/18:0 were higher in the NASH liver of both groups. Saturated sphingomyelins only declined in WT1 NASH liver. Lysophosphatidylcholine concentrations were quite normal in NASH and only one of the 12 altered phosphatidylcholine species declined in NASH liver of both groups. Very few phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol species were comparably regulated in NASH liver of both animal groups. Seven of these lipid species declined and two increased in NASH. Notably, hepatic mRNA expression of proinflammatory (F4/80, CD68, IL-6, TNF and chemerin) and profibrotic genes (TGF beta and alpha SMA) was comparable in WT1 and WT2 mice.ConclusionsMice housed and bred in different animal facilities had comparable disease severity of NASH whereas liver lipids varied among the groups. Thus, there was no specific lipid signature for NASH in the MCD model.

Insight into erythrocyte phospholipid molecular flux in healthy humans and in patients with acute respiratory distress syndrome.

Although the distribution of cellular membrane phospholipid composition is well characterised in human erythrocytes, in-vivo turnover and dynamic flux of phospholipids between plasma and erythrocytes in physiological and in particular during disease states are mostly unknown. Erythrocyte mass primarily consisted of lipids and phosphatidylcholine (PC) contributes to the significant proportion of phospholipid membrane composition. Esterified membrane PC can be utilised during pathological processes to generate pro and anti-inflammatory lipid mediators, which can contribute to the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, utilising isotope labelling of choline and analytical methods with electrospray mass spectrometry (ESI-MS/MS), we characterised individual molecular composition and dynamic exchange of PC, sphingomyelins (SM) and lysophosphatidylcholines (LPC) between plasma and erythrocytes. In ARDS patients, there were significant alterations in PC molecular composition, coupled with a continuous loss of arachidonoyl-PC species over time. Infusion of methyl-D9-choline chloride resulted in enrichment of labelled choline into plasma PC and LPC via CDP-choline pathway with subsequent incorporation into erythrocyte PC. As expected, erythrocyte methyl-D9 PC enrichment is much slower than plasma. Patients had much faster and higher fractional enrichment of all PC and LPC molecules suggesting increased flux between plasma and erythrocytes. There was a particular pattern of incorporation, where the arachidonoyl-PC species achieved equilibrium with plasma rapidly and retained highest concentrations of enrichment compared to the other PC species. Increased enrichment of arachidonoyl-PC coupled with virtually no increase or depletion of its concentrations suggests the possibility of substrate donation for other cell types for the participation of eicosanoid biosynthesis during inflammatory conditions like ARDS. In summary, this study revealed an alerted pattern erythrocyte molecular phospholipid composition and flux in patients with acute respiratory distress syndrome and the pathological consequences of these changes needs further exploration.

Alterations of HDL particle phospholipid composition and role of inflammation in rheumatoid arthritis.

The increased cardiovascular risk in RA (rheumatoid arthritis) cannot be explained by common quantitative circulating lipid parameters. The objective of the study was to characterize the modifications in HDL phosphosphingolipidome in patients with RA to identify qualitative modifications which could better predict the risk for CVD. Nineteen patients with RA were compared to control subjects paired for age, sex, BMI, and criteria of metabolic syndrome. The characterization of total HDL phosphosphingolipidome was performed by LC-MS/MS. RA was associated with an increased HDL content of lysophosphatidylcholine and a decreased content of PC (phosphatidylcholine), respectively, positively and negatively associated with cardiovascular risk. A discriminant molecular signature composed of 18 lipids was obtained in the HDL from RA patients. The detailed analysis of phospholipid species showed that molecules carrying omega-3 FA (fatty acids), notably docosahexaenoic acid (C22:6 n-3), were depleted in HDL isolated from RA patients. By contrast, two PE (phosphatidylethanolamine) species carrying arachidonic acid (C20:4 n-6) were increased in HDL from RA patients. Furthermore, disease activity and severity indexes were associated with altered HDL content of 4 PE and 2 PC species. In conclusion, the composition of HDL phosphosphingolipidome is altered during RA. Identification of a lipidomic signature could therefore represent a promising biomarker for CVD risk. Although a causal link remains to be demonstrated, pharmacological and nutritional interventions targeting the normalization of the FA composition of altered phospholipids could help to fight against RA-related inflammation and CVD risk.

Abstract 885: The Role of Paraoxonase 2 (PON2) in Mitochondrial Membrane Phospholipid Composition and Lipid Peroxidation

Introduction: We have recently reported that PON2 plays a protective role against myocardial ischemia-reperfusion injury (IRI) by reducing oxidative stress and mitochondrial dysfunction, in the form of improved calcium retention capacity and integrity of the mitochondrial membrane potential. It is known that mitochondrial phospholipids (PL) play critical roles in mitochondrial homeostasis via maintenance of normal bioenergetics and membrane function. These PLs undergo oxidative stress during IRI resulting in an increase in lipid peroxidation, damage of the respiratory complexes and loss of mitochondrial membrane potential. Hypothesis: PON2 protects cardiomyocytes against myocardial IRI by modulating the mitochondrial membrane phospholipid composition and lipid peroxidation. Methods: Two methods were developed to measure 1) various oxidized lipid species (i.e. 5, 12, and 15 HETE, 9, 13 HODE, and 5-oxoETE) and inflammatory markers (lipid panel) and 2) a panel of phospholipid species (i.e. PC, PE, PS) (PLP) via mass spectrometry (ESI LC-MS/MS). Mitochondria were isolated from male C57BL6/J (WT) and PON2 deficient (PON2-def) mice. PLs were then extracted using a modified Bligh and Dyer method with the respective internal standards, and run on a SCIEX 5500 QTrap run in negative ion mode and controlled by Analyst 1.6.2 software. Data is expressed as mean±SEM. Student’s T-test is used for statistical analysis, with p&lt;0.05 considered statistically significant. Results: We determined that under baseline conditions, PON2-deficient mice have altered mitochondrial membrane PL composition and lipid peroxidation. We observe that PON2-def mitochondria have a significant decrease in various phosphatidylcholine species and an increase in phosphatidylethanolamine (42:9). In addition, there were variations in the composition of phosphatidylserine and phosphatidylinositol. The alteration of mitochondrial PL were accompanied by significant increases in various oxidized lipids including 9, 13-HODE, 14S, 17S-HDHA, 5, 15-HETE, and 5-oxoETE. Conclusion: Our preliminary studies point to the important role that PON2 plays in modulating mitochondrial PL composition and lipid peroxidation, and warrant further investigation under myocardial IRI.

Abstract 4900: Dynamic metabolic response of prostate cancer patients treated with ADT and low carb diet

Abstract Background: Prostate cancer (PrCa) is one of the most common cancers among men and managed through surgery, hormonal therapy, chemotherapy, radiation, and cryotherapy. Depending on the stage of PrCa, androgen deprivation therapy (ADT) is commonly utilized as an intervention. ADT has proven effective in intervening in PrCa progression, although there are several side effects. Recently, the use of low carb diets has been shown to alter patients’ metabolic phenotype and as such may reduce the side effects of ADT. Methods: In this study 35 men, who were beginning ADT, were randomized to low carb diet intervention with recommended exercise or control (no lifestyle change) for 6 months. Primary results have been reported showing significant weight loss at both 3-month (17lb, p&amp;lt;0.01) and 6-month (23 lbs, p&amp;lt;0.01) and improved insulin control at 3-month (HOMA=-19 as compared to baseline, p=0.02) but not at 6-month (HOMA=-4 as compared to baseline, p=0.13). For this exploratory analysis, sera collected at baseline, 3, and 6 months was used for metabolomics analysis utilizing GC/MS TOF, QqQ LC-HILIC-MS/MS, and TripleTOF 6600 LC-RP-MS and lipidomics analysis using TripleTOF 5600+ MS/MSALL workflows to quantify the chemical diversity of the metabolome and lipidome. Results: Over 450 metabolites and 1000 lipid species were quantified. Metabolomics analysis of sera from the control patients (ADT alone) demonstrated alterations in steroid biosynthesis, androgen/estrogen metabolism, fatty acid metabolism, and lysine degradation. Lipidomics analysis of the control patients demonstrated changes in selective long chain polyunsaturated phosphatidylcholine species. Analysis of the ADT plus low carb patients revealed a change in pyruvate, glucose-alanine cycle, selenoamino acid, phenylalanine/tyrosine, and taurine metabolism. Further, lipidomic analysis revealed changes in several triglycerides, plasmalenylethanolamine, and phosphatidic acid species. Conclusion: In summary, integration of metabolomic and lipidomic analysis in hormonal and metabolic interventions of PrCa patients revealed dynamic changes providing novel insight for tailoring therapeutic intervention and monitoring. Citation Format: Stephen Freedland, Pao-Hwa Lin, Emily Y. Chen, Vladimir Tolstikov, Jen-Tsan Chi, Rangaprasad Sarangarajan, Niven R. Narain, Michael A. Kiebish. Dynamic metabolic response of prostate cancer patients treated with ADT and low carb diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4900.

Lipid Isolation Process and Study on Some Molecular Species of Polar Lipid Isolated from Seed of Madhuca ellitica

This study attempted the lipid extraction process from the seeds of Madhuca ellitica, a lipid-rich plant, and conducted a lipidomic analysis on molecular species of the obtained product. Total lipids of the crude seeds were found to contain 11.2% of polar lipids. The major fatty acids (FAs) of the polar lipids were palmitic (16:0), stearic (18:0), oleic (18:1n-9), and linoleic (18:2n-6) acids, which amounted to 28.5, 12.5, 44.8, and 13.2% of total FAs, respectively. The content and chemical structures of individual molecular species of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), and sulfoquinovosyldiacylglycerol (SQDG) were determined by HPLC with a tandem high-resolution mass spectrometry (HRMS). The major molecular species were 18:1/18:2 PE, 16:0/18:1 PC, 18:1/18:2 PC, 16:0/18:2 PG, 16:0/18:1 PG, 16:1/18:1 PI, 16:0/18:1 PI, 18:0/18:2 PI, 16:0/18:1 PA, 18:1/18:2 PA, 16:0/18:1 SQDG, and 18:0/18:1 SQDG. The application of a tandem HRMS allows us to determine the content of each isomer in pairs of the monoisotopic molecular species, for example, 18:0/18:2 and 18:1/18:1. The evaluation of the seed polar lipid profile will be helpful for developing the potential of this tree for nutritive and industrial uses.

Childhood growth and neurocognition are associated with distinct sets of metabolites

BackgroundUndernutrition is a serious global problem that contributes to increased child morbidity and mortality, impaired neurocognitive development, and decreased educational and economic attainment. Current interventions are only marginally effective, and identification of associated metabolic pathways can offer new strategies for intervention. MethodsPlasma samples were collected at 9 and 36 months from a subset of the PROVIDE child cohort (n = 130). Targeted metabolomics was performed on bile acids, acylcarnitines, amino acids, phosphatidylcholines, and sphingomyelins. Metabolic associations with linear growth and neurocognitive outcomes at four years were evaluated using correlation and penalized-linear regression analysis as well as conditional random forest modeling. FindingsDifferent metabolites were associated with growth and neurocognitive outcomes. Improved growth outcomes were associated with higher concentrations of hydroxy-sphingomyelin and essential amino acids and lower levels of acylcarnitines and bile acid conjugation. Neurocognitive scores were largely associated with phosphatidylcholine species and early metabolic indicators of inflammation. All metabolites identified explain ~45% of growth and neurocognitive variation. InterpretationGrowth outcomes were predominantly associated with metabolites measured early in life (9 months), many of which were biomarkers of insufficient diet, environmental enteric dysfunction, and microbiome disruption. Hydroxy-sphingomyelin was a significant predictor of improved growth. Neurocognitive outcome was predominantly associated with 36 month phosphatidylcholines and inflammatory metabolites, which may serve as important biomarkers of optimal neurodevelopment. The distinct sets of metabolites associated with growth and neurocognition suggest that intervention may require targeted approaches towards distinct metabolic pathways. FundBill & Melinda Gates Foundation (OP1173478); National Institutes of Health (AI043596, CA044579).

The activity of the saponin ginsenoside Rh2 is enhanced by the interaction with membrane sphingomyelin but depressed by cholesterol

The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts with our recent publication showing that Chol, contrary to sphingomyelin (SM), can delay the cytotoxicity of the saponin ginsenoside Rh2, challenging the usual view that most saponins mediate their membrane effects through interaction with Chol. The aim of the present study was to elucidate the respective importance of Chol and SM as compared to phosphatidylcholine (PC) species in the membrane-related effects of Rh2. On simple lipid monolayers, Rh2 interacted more favorably with eggSM and DOPC than with Chol and eggPC. Using Large Unilamellar Vesicles (LUVs) of binary or ternary lipid compositions, we showed that Rh2 increased vesicle size, decreased membrane fluidity and induced membrane permeability with the following preference: eggSM:eggPC > eggSM:eggPC:Chol > eggPC:Chol. On Giant Unilamellar Vesicles (GUVs), we evidenced that Rh2 generated positive curvatures in eggSM-containing GUVs and small buds followed by intra-luminal vesicles in eggSM-free GUVs. Altogether, our data indicate that eggSM promotes and accelerates membrane-related effects induced by Rh2 whereas Chol slows down and depresses these effects. This study reconsiders the theory that Chol is the only responsible for the activity of saponins.

Enhanced Fatty Acid Scavenging and Glycerophospholipid Metabolism Accompany Melanocyte Neoplasia Progression in Zebrafish.

Alterations in lipid metabolism in cancer cells impact cell structure, signaling, and energy metabolism, making lipid metabolism a potential diagnostic marker and therapeutic target. In this study, we combined PET, desorption electrospray ionization-mass spectrometry (DESI-MS), nonimaging MS, and transcriptomic analyses to interrogate changes in lipid metabolism in a transgenic zebrafish model of oncogenic RAS-driven melanocyte neoplasia progression. Exogenous fatty acid uptake was detected in melanoma tumor nodules by PET using the palmitic acid surrogate tracer 14(R,S)-18F-fluoro-6-thia-heptadecanoic acid ([18F]-FTHA), consistent with upregulation of genes associated with fatty acid uptake found through microarray analysis. DESI-MS imaging revealed that FTHA uptake in tumors was heterogeneous. Transcriptome and lipidome analyses further highlighted dysregulation of glycerophospholipid pathways in melanoma tumor nodules, including increased abundance of phosphatidyl ethanolamine and phosphatidyl choline species, corroborated by DESI-MS, which again revealed heterogeneous phospholipid composition in tumors. Overexpression of the gene encoding lipoprotein lipase (LPL), which was upregulated in zebrafish melanocyte tumor nodules and expressed in the majority of human melanomas, accelerated progression of oncogenic RAS-driven melanocyte neoplasia in zebrafish. Depletion or antagonism of LPL suppressed human melanoma cell growth; this required simultaneous fatty acid synthase (FASN) inhibition when FASN expression was also elevated. Collectively, our findings implicate fatty acid acquisition as a possible therapeutic target in melanoma, and the methods we developed for monitoring fatty acid uptake have potential for diagnosis, patient stratification, and monitoring pharmacologic response. SIGNIFICANCE: These findings demonstrate the translational potential of monitoring fatty acid uptake and identify lipoprotein lipase as a potential therapeutic target in melanoma.

The acyltransferase Gpc1 impacts PC molecular species and phenotypic outcomes

Phospholipids are major components of cellular membranes and their biochemical properties, such as fatty acid composition, help define the biophysical properties of membranes. Phosphatidylcholine (PC) is the most abundant phospholipid in cellular membranes and is synthesized primarily by two well defined metabolic pathways in Saccharomyces cerevisiae. Our laboratory recently characterized a novel glycerophosphocholine (GPC) acyltransferase, encoded by GPC1, that provides a third route for PC biosynthesis and is involved in the newly defined PC deacylation‐reacylation remodeling pathway (PC‐DRP). PC‐DRP begins with GPC, the product of complete PC deacylation by phospholipases of the B type. Gpc1 acylates GPC to form lysophosphatidylcholine (LysoPC). LysoPC can subsequently be converted to PC by Ale1. Previous results have shown that Gpc1 prefers the addition of saturated fatty acids during PC remodeling, which results in more saturated PC species and has the potential to influence the biophysical properties of the membrane. To better understand how GPC1 is regulated and to determine its physiological role in the cell, we assessed the effect of GPC1 dosage and temperature alterations on growth, expression, and PC molecular species. Preliminary data indicates that while increasing the temperature from 30°C to 37°C slows growth in a wild type strain, the gpc1Δ mutant is much less effected. As expected, overexpression of GPC1 further negatively impacts growth at 37°C. GPC1 message abundance, as measured by qRT‐PCR, is decreased at 37°C as compared to 30°C in a wild type strain. Other members of PC‐DRP, including ALE1 and PLB1 also display decreased expression upon elevated temperature. Mass spectrometry‐based analysis of the PC molecular species under these conditions are ongoing. Other stressors expected to impact membrane events, including inositol availability, are being examined. These experiments will provide insight into the physiological role of Gpc1.Support or Funding InformationNIH R15 GM104876 (to JPV)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Production of Matchout-Deuterated Cholesterol and the Study of Bilayer-Cholesterol Interactions

The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron scattering and neutron reflection, the level of deuteration of a sample can be varied to match the scattering length density of a specific D2O/H2O solvent mixture. This can be of major value in structural studies where specific regions of a complex system can be highlighted, and others rendered invisible. This is especially useful in analyses of the structure and dynamics of membrane components. In mammalian membranes, the presence of cholesterol is crucial in modulating the properties of lipids and in their interaction with proteins. Here, a protocol is described for the production of partially deuterated cholesterol which has a neutron scattering length density that matches that of 100% D2O solvent (hereby named matchout cholesterol). The level of deuteration was determined by mass spectrometry and nuclear magnetic resonance. The cholesterol match-point was verified experimentally using small angle neutron scattering. The matchout cholesterol was used to investigate the incorporation of cholesterol in various phosphatidylcholine supported lipid bilayers by neutron reflectometry. The study included both saturated and unsaturated lipids, as well as lipids with varying chain lengths. It was found that cholesterol is distributed asymmetrically within the bilayer, positioned closer to the headgroups of the lipids than to the middle of the tail core, regardless of the phosphatidylcholine species.

Phospholipid profiles and hepatocellular carcinoma risk and prognosis in cirrhotic patients.

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Phospholipids are now well-recognised players in tumour progression. Their metabolic tissue alterations can be associated with plasmatic modifications. The aim of this study was to evaluate the potential of the plasma phospholipid profile as a risk and prognostic biomarker in HCC.MethodsNinety cirrhotic patients with (cases) or without HCC (controls) were studied after matching for inclusion centre, age, gender, virus infection, cirrhosis duration and Child-Pugh grade. High-performance liquid chromatography coupled with tandem-mass spectrometry was used to quantify the main species of seven categories of phospholipids in plasma.ResultsElevated concentrations of phosphatidylcholine (PC) 16:0/16:1 (p=0.0180), PC 16:0/16:0 (p=0.0327), PC 16:0/18:1 (p=0.0264) and sphingomyelin (SM) 18:2/24:1 (p=0.0379) and low concentrations of lysophosphatidylcholine 20:4 (0.0093) and plasmalogen-phosphatidylethanolamine (pPE) 16:0/20:4 (p=0.0463), pPE 18:0/20:4 (p=0.0077), pPE 18:0/20:5 (p=0.0163), pPE 18:0/20:3 (p=0.0463) discriminated HCC patients from cirrhotic controls. Two ceramide species were associated with increased HCC risk of death while lysophospholipids, a polyunsaturated phosphatidylinositol, some PC and SM species were associated with low risk of death in HCC patients in 1 and/or 3 years.ConclusionThis study identified phospholipid profiles related to HCC risk in liver cirrhotic patients and showed for the first time the potential of some phospholipids in predicting HCC patient mortality.

Combined choline and DHA supplementation: a randomized controlled trial.

Choline and docosahexaenoic acid (DHA) are essential nutrients for preterm infant development. They are metabolically linked via phosphatidylcholine (PC), a constitutive plasma membrane lipid and the major transport form of DHA in plasma. Plasma choline and DHA-PC concentrations rapidly decline after preterm birth. To improve preterm infant nutrition, we evaluated combined compared to exclusive choline and DHA supplementation, and standard feeding. Randomized partially blinded single-center trial. Neonatal tertiary referral center in Tübingen, Germany. 24 inborn preterm infants < 32 week postmenstrual age. Standard nutrition (control) or, additionally, enteral choline (30mg/kg/day), DHA (60mg/kg/day), or both for 10days. Single enteral administration of 3.6mg/kg [methyl-D9-] choline chloride as a tracer at 7.5days. Primary outcome variable was plasma choline following 7 days of supplementation. Deuterated and unlabeled choline metabolites, DHA-PC, and other PC species were secondary outcome variables. Choline supplementation increased plasma choline to near-fetal concentrations [35.4 (32.8-41.7)µmol/L vs. 17.8 (16.1-22.4) µmol/L, p < 0.01] and decreased D9-choline enrichment of PC. Single DHA treatment decreased DHA in PC relative to total lipid [66 (60-68)% vs. 78 (74-80)%; p < 0.01], which was prevented by choline. DHA alone increased DHA-PC only by 35 (26-45)%, but combined treatment by 63 (49-74)% (p < 0.001). D9-choline enrichment showed preferential synthesis of PC containing linoleic acid. PC synthesis via phosphatidylethanolamine methylation resulted in preferential synthesis of DHA-containing D3-PC, which was increased by choline supplementation. 30mg/kg/day additional choline supplementation increases plasma choline to near-fetal concentrations, dilutes the D9-choline tracer via increased precursor concentrations and improves DHA homeostasis in preterm infants. clinicaltrials.gov. Identifier: NCT02509728.

The Fatty Acid Composition of Major Membrane Lipids of the Mussel Crenomytilus grayanus (Dunker, 1853) (Bivalvia: Mytilidae) under Chronic Anthropogenic Pollution: Evaluation of Stability

Accumulation of heavy metals and their effect on the composition of fatty acids and molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) has been studied in the molluskan organs and tissues of the Gray mussel from clean and polluted water areas of Peter the Great Bay of the Sea of Japan. The total load of heavy metals accumulated in the mussel was evaluated with the coefficient of total concentration (ΣCC), which turned out to be extremely high for all the organs studied in mollusks from a polluted site. Cu and Pb accounted for the main share in the total load. It was found that accumulation of heavy metals in the kidneys, gills, and muscles had no effect on the composition of fatty acids, molecular species, and forms of basic membrane lipids of PC and PE. It has been shown that high resistance of membrane lipids of the Gray mussel to chronic anthropogenic pollution with heavy metals allows the mussel to function under conditions of contamination.

Distinct phospholipid and sphingolipid species are linked to altered HDL function in apolipoprotein A-I deficiency

Familial apolipoprotein A-I (apoA-I) deficiency (FAID) involving low levels of both apoA-I and high-density lipoprotein (HDL) cholesterol is associated with accelerated atherosclerosis. The objective of this study was to define distinctive patterns in the lipidome of HDL subpopulations in FAID in relationship to antiatherogenic activities. Five HDL subfractions were isolated by ultracentrifugation from plasma of FAID Caucasian patients (n=5) and age-matched healthy normolipidemic Caucasian controls (n=8), and the HDL lipidome (160 molecular species of 9 classes of phospholipids and sphingolipids) was quantitatively evaluated. Increased concentrations of numerous molecular species of lysophosphatidylcholine (up to 12-fold), ceramides (up to 3-fold), phosphatidylserine (up to 34-fold), phosphatidic acid (up to 71-fold), and phosphatidylglycerol (up to 20-fold) were detected throughout all five HDL subpopulations as compared with their counterparts from controls, whereas concentrations of phosphatidylethanolamine species were decreased (up to 5-fold). Moderately to highly abundant, within their lipid class, species of phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and ceramide featuring multiple unsaturations were primarily affected by apoA-I deficiency; their HDL content, particularly that of phosphatidylcholine (34:2), was strongly correlated with HDL function, impaired in FAID. Metabolic pathway analysis revealed that sphingolipid, glycerophospholipid, and linoleic acid metabolism was significantly affected by FAID. These data reveal that altered content of specific phospholipid and sphingolipid species is linked to deficient antiatherogenic properties of HDL in FAID.

Hepatic stellate cell activation: A source for bioactive lipids

Hepatic stellate cells (HSCs) are non-parenchymal liver cells that characteristically contain multiple retinoid (vitamin A)-containing lipid droplets. In this study, we addressed the metabolic fate of non-retinoid lipids originating from lipid droplet loss during HSCs activation. UPLC/MS/MS and qRT-PCR were used to monitor the lipid composition and mRNA expression of selected genes regulating lipid metabolism in freshly isolated, overnight-, 3- and 7-day cultures or primary mouse HSCs. A preferential accumulation of specific C20-C24 fatty acid species, especially arachidonic (C20:4) and docosahexaenoic acids (C22:6), was revealed in culture-activated HSCs along with an upregulation of transcription of fatty acid desaturases (Scd1, Scd2) and elongases (Elovl5, Elovl6). This was accompanied with an enrichment of activated HSCs with 36:4 and 38:4 phosphatidylcholine species containing polyunsaturated fatty acids and associated accumulation of selective lipid mediators, including endocannabinoids and related N-acylethanolamides, as well as ceramides. An increase in 2-arachidonoylglycerol and N-arachydonoylethanolamide concentrations was observed along with an upregulation of Daglα mRNA expression in HSCs during culture activation. N-palmitoylethanolamide was identified as the most abundant endocannabinoid-like species in activated HSCs. An increase in total ceramide levels and enrichment with N-palmitoyl (C16:0), N-tetracosenoyl (C24:1), N-tetracosanoyl (C24:0) and N-docosanoyl (C22:0) ceramides was detected in activated HSC cultures and was preceded by increased mRNA expression of ceramide synthesizing enzymes (CerS2, CerS5 and Smpd1). Our data suggest an active redistribution of non-retinoid lipids in HSCs underlying the formation of low abundance, highly bioactive lipid species that may affect signaling during HSC activation, as well as extracellularly within the liver.