Discovery of Unexpected Sphingolipids in Almonds and Pistachios with an Innovative Use of Triple Quadrupole Tandem Mass Spectrometry

The densely packed storage of valuable nutrients (carbohydrates, lipids, proteins, micronutrients) in the endosperm of nuts and seeds makes the study of their complex composition a topic of great importance. Ceramides in the total lipid extract of some ground almonds and pistachios were searched with a systematic innovative discovery precursor ion scan in a triple quadrupole tandem mass spectrometry, where iso-energetic collision activated dissociation was performed. Five descriptors were used to search components with different C18 long chain bases containing different structural motifs (d18:0, d18:1, d18:2, t18:0, t18:1). The presence of hexoside unit was screened with a specific neutral loss experiment under iso-energetic collision activated dissociation conditions. The discovery scans highlighted the presence of two specific hexosyl-ceramides with a modified sphingosine component (d18:2) and C16:0 or C16:0 hydroxy-fatty acids. The hexosyl-ceramide with the non-hydroxylated fatty acid seemed specific of pistachios and was undetected in almonds. The fast and comprehensive mass spectrometric method used here can be useful to screen lipid extracts of several more seeds of nutraceutical interest, searching for unusual and/or specific sphingosides with chemically decorated long chain bases.

Adipose tissue fatty acid and lipid mediator composition in obesity and response to chronic marine omega-3 fatty acid supplementation

Introduction:Obesity is an excess of adipose tissue (AT) and is linked with increased inflammation that enhances risk of type-2 diabetes and cardiovascular disease. The BIOCLAIMS study assessed the fatty acid (FA) and lipid mediator composition in normal weight and obese individuals, and their response to chronic omega-3 FA supplementation.Materials and methods:AT biopsies were collected pre- and post-12 week supplementation with 1.1 g EPA + 0.8 g DHA/day or corn oil. The composition of FA in the total lipid extract (TLE) of AT from 37 normal weight and 44 obese subjects was assessed by gas chromatography (GC) and the concentration of lipid mediators in AT TLE of 36 normal weight and 45 obese subjects by coupled GC-mass spectrometry.Results:AT of obese subjects had higher concentrations of arachidonic acid (AA), EPA, DPA, PGF2α, arachidonoylethanolamine (AEA), and eicosapentaenoylethanolamine (EPEA) than that of normal weight subjects (P < 0.05). Obese subjects also had and lower concentrations of mediators derived from linoleic and α-linolenic acids, DHA-derived mediators including RvD2 and hydroxydocosahexaenoic acids (HDHAs), AA-derived mediators including lipoxin-B4, hepoxilin-A3, 11,12-dihydroxyeicosatetrienoic acid (DHET), and 6-keto-PGF1α, and the FA ethanolamine of myristic acid and glycerol ester of palmitic acid (P < 0.05 all) than normal weight subjects.Chronic supplementation with EPA + DHA increased the concentration of EPA (P = 0.006) and EPA-derived lipid mediators including dihydroxyeicosatetrienoic acids, EPEA, and EPA-glycerol ester (P < 0.05), and increased the concentration of DHA (P < 0.001), docosahexaenoylethanolamine, and 8-, 11-, 14-, and 16-HDHA (P < 0.05). Chronic supplementation with EPA + DHA also decreased the concentration 4-, 17-, and 20-HDHA, and AA-derived lipid mediators including DHETs, AEA, and LTs in normal weight subjects (P < 0.05), and decreased 2-AG in obese subjects (P < 0.05).Discussion:These data indicate altered lipid signalling in AT in obesity (at baseline) suggesting dysregulation of adipose tissue expansion and inflammatory signalling, including lack of self-resolution, as well as dysregulation in the utilization of supplementary EPA + DHA for synthesis of anti-inflammatory lipid mediators. EPA + DHA are able to modulate synthesis of EPA-, DHA- and AA-derived lipid mediators but obesity may involve resistance to these effects particularly in endocannabinoid synthesis.

Glycerolipid Composition of the Red Macroalga Agarophyton Chilensis and Comparison to the Closely Related Agarophyton Vermiculophyllum Producing Different Types of Eicosanoids

The red macroalga Agarophyton chilensis is a well-known producer of eicosanoids such as hydroxyeicosatetraenoic acids, but the alga produces almost no prostaglandins, unlike the closely related A. vermiculophyllum. This indicates that the related two algae would have different enzyme systems or substrate composition. To carry out more in-depth discussions on the metabolic pathway of eicosanoids between the two algae, we investigated the characteristics of glycerolipids, which are the substrates of eicosanoids production, of A. chilensis and compared them to the reported values of A. vermiculophyllum. In A. chilensis, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylcholine (PC) were the major lipid classes and accounted for 44.4% of the total lipid extract. The predominant fatty acids were arachidonic acid (20:4n-6), an eicosanoids precursor, and palmitic acid (16:0). The 20:4n-6 content was extremely high in MGDG and PC (>70%), and the 16:0 content was extremely high in DGDG and SQDG (>40%). A chiral-phase HPLC analysis showed that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The glycerolipid molecular species were determined by reversed-phase HPLC–ESI–MS analysis. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (63.8%) and PC (48.2%), 20:4n-6/16:0 for DGDG (71.1%) and SQDG (29.4%). These lipid characteristics of A. chilensis were almost the same as those of A. vermiculophyllum. Hence, the differences of the eicosanoids producing ability between the two algae would not be due to the difference of substrate composition but the difference of enzyme system.

A Liposomal Platform for Sensing of Extracellular Analytes Near Cells

Cell-permeable fluorescent chemosensors (calcein, monochlorobimane, and a recently reported spiropyran-based sensor SP2) have been incorporated into yeast total lipid extract-based liposomes to suppress inherent cell permeability to allow the detection of extracellular Ca2+, GSH, and Zn2+, respectively. The repurposed sensors have enhanced aqueous solubility and the ability to quantitatively measure biologically relevant concentrations of Ca2+ (0.25 mM–1 mM), Zn2+ (6.25 µM–50 µM), and GSH (0.25 mM–1 mM) by fluorescence in aqueous media. In addition, the liposomal sensors are nontoxic to HEK293 cells and have the ability to detect exogenously added Zn2+ (1 mM), Ca2+ (1 mM), or GSH (1 mM) near cells without internalisation. This new sensing platform provides a means to repurpose a range of intracellular fluorescent sensors to specifically detect extracellular analytes, while also improving biocompatibility for overall enhanced use in a wide range of biomedical applications.

Model Neural Membrane Droplet Interface Bilayers from Brain Total Lipid Extract for Studying Membrane-Peptide Interactions with Amyloid-β

ABSTRACTDroplet interface bilayers (DIBs) are formed using brain total lipid extract (BTLE) to create a synthetic bilayer whose lipid composition mimics that of neural cells. The electrical properties of BTLE DIBs, specifically membrane resistance, capacitance, and rupture potential, are determined and compared to the properties of bilayers formed using DPhPC, the most common lipid within the growing DIB field. There is no significant difference in the resistance or rupture potential of BTLE and DPhPC bilayers, for instance with average nominal resistance over 200 GΩ and rupture potential around 200 mV. In electrical measurements with either DPhPC or BTLE bilayers, applied voltages of up to ±150 mV yield low levels of leakage current. Upon interaction with the pore-forming amyloid-beta (Aβ) peptide, both bilayers display sudden significant voltage-dependent increases in conductance with characteristic threshold voltages well below 150 mV. Discrete single-channel type events are observed in the case of Aβ-BTLE whereas disordered fluctuating conductance is observed with Aβ-DPhPC. Circular dichroism is measured for Aβ incubated with BTLE and DPhPC liposomes, as well as pure Aβ, at a range of temperatures over a period of several weeks. Changes in secondary structure of liposome-bound and pure Aβ are significantly affected by both lipid type and temperature. A key finding includes the 100% conversion of Aβ to alpha-helical confirmation within 24 hours when incubated with liposomes (of either type) at physiologically relevant 37°C. The 100% alpha-helical Aβ is maintained for up to 2 weeks at 37°C when incubated with liposomes, although other structures begin to emerge after the 14 day mark. Between 14-31 days after reconstitution, Aβ incubated at 37C with BTLE bilayers displays longer lasting alpha-helical content than DPhPC. At the same temperature, pure Aβ is 100% alpha helical only at the 1 day mark with apparent restructuring from day 2 through day 31. Refrigerated Ab samples do not display 100% alpha-helical structure across the entire 31 day testing period. The differences observed between BTLE and DPhPC in both electrophysiological and spectroscopic experiments may be a result of phase separations or other variations in membrane fluidity that result from the use of a homogeneous total lipid extract. Time and temperature play essential roles in the aggregation and restructuring of potentially toxic Aβ oligomers, and there is motivation for further efforts to elicit the mechanistic differences in interactions of Ab with BTLE compared to DPhPC.

Thermoflavifilum aggregans gen. nov., sp. nov., a thermophilic and slightly halophilic filamentous bacterium from the phylum Bacteroidetes

A strictly aerobic, thermophilic, moderately acidophilic, non-spore-forming bacterium, strain P373T, was isolated from geothermally heated soil at Waikite, New Zealand. Cells were filamentous rods, 0.2–0.4 µm in diameter and grew in chains up to 80 µm in length. On the basis of 16S rRNA gene sequence similarity, strain P373T was shown to belong to the family Chitinophagaceae (class Sphingobacteriia ) of the phylum Bacteroidetes , with the most closely related cultivated strain, Chitinophaga pinensis UQM 2034T, having 87.6 % sequence similarity. Cells stained Gram-negative, and were catalase- and oxidase-positive. The major fatty acids were i-15 : 0 (10.8 %), i-17 : 0 (24.5 %) and i-17 : 0 3-OH (35.2 %). Primary lipids were phosphatidylethanolamine, two unidentified aminolipids and three other unidentified polar lipids. The presence of sulfonolipids (N-acyl-capnines) was observed in the total lipid extract by mass spectrometry. The G+C content of the genomic DNA was 47.3 mol% and the primary respiratory quinone was MK-7. Strain P373T grew at 35–63 °C with an optimum temperature of 60 °C, and at pH 5.5–8.7 with an optimum growth pH of 7.3–7.4. NaCl tolerance was up to 5 % (w/v) with an optimum of 0.1–0.25 % (w/v). Cell colonies were non-translucent and pigmented vivid yellow–orange. Cells displayed an oxidative chemoheterotrophic metabolism. The distinct phylogenetic position and the phenotypic characteristics separate strain P373T from all other members of the phylum Bacteroidetes and indicate that it represents a novel species in a new genus, for which the name Thermoflavifilum aggregans gen. nov., sp. nov. is proposed. The type strain of the type species is P373T ( = ICMP 20041T = DSM 27268T).