Phosphatidylserine from Portunus trituberculatus Eggs Alleviates Insulin Resistance and Alters the Gut Microbiota in High-Fat-Diet-Fed Mice.

Portunus trituberculatus eggs contain phospholipids, whose components and bioactivity are unclear. Here, we investigated the fatty acid composition of phosphatidylserine from P. trituberculatus eggs (Pt-PS). Moreover, its effects on insulin resistance and gut microbiota were also evaluated in high-fat-diet-fed mice. Our results showed that Pt-PS accounted for 26.51% of phospholipids and contained abundant polyunsaturated fatty acids (more than 50% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)). Animal experiments indicated that Pt-PS significantly decreased body weight and adipose weight gain, improved hyperglycemia and hyperinsulinemia, mitigated insulin resistance, and regulated circulatory cytokines. Pt-PS activated insulin receptor substrate 1 (IRS1) and increased the levels of IRS1-associated phosphatidylinositol 3-hydroxy kinase (PI3K), phosphorylated protein kinase B (Akt) protein, and plasma membrane glucose transporter 4 protein. Furthermore, Pt-PS modified the gut microbiota, inducing, especially, a dramatic decrease in the ratio of Firmicutes to Bacteroidetes at the phylum level, as well as a remarkable improvement in their subordinate categories. Pt-PS also reduced fecal lipopolysaccharide concentration and enhanced fecal acetate, propionate, and butyrate concentrations. Additionally, the effects of Pt-PS on alleviation of insulin resistance and regulation of intestinal bacteria were better than those of phosphatidylserine from soybean. These results suggest that Pt-PS mitigates insulin resistance by altering the gut microbiota. Therefore, Pt-PS may be developed as an effective food supplement for the inhibition of insulin resistance and the regulation of human gut health.

A comparison between continuous and batch processes to produce phosphatidylserine in the efficient and green aqueous-solid system.

The efficient and environmentally friendly aqueous-solid systems employed Triton-X-100-modified silica as the "artificial interface" to adsorb phosphatidylcholine (PC) in purely aqueous solutions and silica-adsorbed PC was successfully used for phospholipase D (PLD)-mediated transphosphatidylation. Three kinds of silicas with different sizes were employed to investigate advantages and disadvantages of batch and continuous technologies for PLD-catalyzed transphosphatidylation in aqueous-solid systems. The highest yields of product were obtained in the batch technology, but the continuous production had the simplest operational process and highest space-time yield. After transphosphatidylation, the product adsorbed on carriers were eluted by coconut oil and used to manufacture relevant hard, soft, and micro-capsules. Special attention has been paid to the preparation of microcapsules. Toxic solvents were completely avoided in the whole technological process including production and product packaging. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2777, 2019.

High-Yield and Sustainable Production of Phosphatidylserine in Purely Aqueous Solutions via Adsorption of Phosphatidylcholine on Triton-X-100-Modified Silica.

Triton X-100 was covalently bound to a surface of silica and acted as an anchor molecule to facilitate the adsorption of phosphatidylcholine (PC) in a purely aqueous solution. The silica-adsorbed PC obtained was successfully used for phospholipase D (PLD)-mediated transphosphatidylation in the production of phosphatidylserine (PS). Organic solvents were completely avoided in the whole production process. The PC loading and PS yield reached 98.9 and 99.0%, respectively. Two adsorption models were studied, and the relevant parameters were calculated to help us understand the adsorption and reaction processes deeply. In addition, the silica-adsorbed PC provides a promising way to continuously biosynthesize PS. A packed-bed reactor was employed to demonstrate the process flow of the continuous production of PS. The recyclability and stability of the Triton-X-100-modified silica were excellent, as demonstrated by its use 30 times during continuous operation without any loss of the productivity.

Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes.

Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

Regional changes in CNS and retinal glycerophospholipid profiles with age: a molecular blueprint.

We present here a quantitative molecular blueprint of the three major glycerophospholipid (GPL) classes, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), in retina and six regions of the brain in C57Bl6 mice at 2, 10, and 26 months of age. We found an age-related increase in molecular species containing saturated and monoenoic FAs and an overall decrease in the longer-chain PUFA molecular species across brain regions, with loss of DHA-containing molecular species as the most consistent and dramatic finding. Although we found very-long-chain PUFAs (VLC-PUFAs) (C28) in PC in the retina, no detectable levels were found in any brain region at any of the ages examined. All brain regions (except hippocampus and retina) showed a significant increase with age in PE plasmalogens. All three retina GPLs had di-PUFA molecular species (predominantly 44:12), which were most abundant in PS (∼30%). In contrast, low levels of di-PUFA GPL (1-2%) were found in all regions of the brain. This study provides a regional and age-related assessment of the brain's lipidome with a level of detail, inclusion, and quantification that has not heretofore been published.

Mass spectrometric analyses of phospholipids in the S334ter-3 rat model of retinal degeneration.

PURPOSE: The purpose of this study was to profile the endogenous phospholipid species in the retinal tissue of the S334ter-3 rat model of retinal degeneration. Retinal tissue was collected from S334ter-3 rats at postnatal day (P) 20, P30, and P60, while control retinal samples were collected from Sprague-Dawley (SD) rats at the same time points for comparison. METHODS: Lipids were extracted using the Bligh and Dyer method, and resuspended in an acetonitrile/isopropanol (1:1) solution. For lipid analyses, a positive ion-mode precursor ion scan (PIS) was used for phosphatidylcholine (PC; product m/z of 184), a negative ion-mode neutral loss scan (NLS) was used for phosphatidylserine (PS; product m/z of 87.1), and a negative ion-mode PIS was used for phosphatidylinositol (PI; product m/z of 241) and phosphatidylethanolamine (PE; product m/z of 196); the analyses were carried out using a TSQ Quantum Access Max mass spectrometer. The samples were directly infused with a Triversa Nanomate using 1.6 kV and 0.4 psi of pressure for the positive ion mode, and 1.3 kV and 0.6 psi of pressure for the negative ion mode, and scanned for 2 min between 200 m/z and 1000 m/z. Ratiometric quantification was performed using quantitative standards for each lipid class. RESULTS: The comparative profiles of PC, PE, PS, and PI between S334ter-3 and control rats showed that there were several lipid species common to both groups, as well as several that were unique to the S334ter-3 group and vice versa. CONCLUSIONS: It was found that the proportions of PC and PS were higher in the control retina compared to S334ter-3, and that the proportions of PE and PI were higher in the S334ter-3 retina compared to control.

The use of a new, modified Dittmer-Lester spray reagent for phospholipid determination by the TLC image analysis technique.

A new phospholipid-specific spray reagent is described. A new phospholipid-specific spray reagent, which is a modification of the Dittmer-Lester reagent, is described in authors' studies. The difference between these two reagents is in the addition of tin (II) chloride to the proposed spray reagent. The use of the described spray reagent together with an image analysis technique allows not only for qualitative, but also for quantitative, determination of major phospholipid classes. Separation of phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was conducted on an HPTLC plate with a mixture of chloroform, methanol and 25% ammonia solution in a volume ratio of 65:25:4 as mobile phase. The calibration curves were linear in the ranges of 5.0-25.0, 1.5-15.0 and 1.0-20.0 µg/spot for PC, PS and PE, respectively. The use of the new spray reagent resulted also in lower limits of detection than the standard molybdenum method for the investigated phospholipids. The proposed method was used to determine the amount of PS in the dietary supplement 'Session', and of PS, PE and PC in biological samples, with good results.

The change of cellular membranes on apoptosis: fluorescence detection.

The strong plasma membrane asymmetry existing in living cells is lost on apoptosis, and it is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the labeled annexin V is used for its detection. The requirement for early and Ca(2+)-independent detection of apoptosis in the formats of spectroscopy of cell suspensions, flow cytometry, microarray technology and confocal or two-photon microscopy stimulated efforts for the development of new methods. Since the PS exposure must produce integrated changes of electrostatic potential and hydration in the outer leaflet of cell membrane, its detection can be provided by direct response of smart fluorescence probes. This review is focused on basic mechanisms underlying the loss of membrane asymmetry during apoptosis and the principles lying in the background of new methods that demonstrate essential advantages over the annexin V-binding assay. The convenient wavelength-ratiometric technique based on fluorescent probe F2N12S is described in detail. It incorporates spontaneously into outer leaflet of cell membrane and the color change of its fluorescent emission associated with apoptosis can be easily detected. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Structural elucidation of molecular species of pacific oyster ether amino phospholipids by normal-phase liquid chromatography/negative-ion electrospray ionization and quadrupole/multiple-stage linear ion-trap mass spectrometry.

Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.

The effect of phosphatidylserine-containing omega-3 fatty acids on memory abilities in subjects with subjective memory complaints: a pilot study.

OBJECTIVE: To evaluate for the first time the efficacy of safe-sourced phosphatidylserine-containing omega-3 long chain polyunsaturated fatty acid (PS-omega-3) in improving memory abilities. METHODS: PS-omega-3 was administered daily for 6 weeks to eight elderly volunteers with subjective memory complaints. The Cognitive Drug Research test battery was used to assess the effect on their cognitive abilities. RESULTS: PS-omega-3 supplementation resulted in 42% increase in the ability to recall words in the delayed condition. CONCLUSION: PS-omega-3 may have a favorable effect on memory in subjects with subjective memory complaints. PS-omega-3 may serve as a safe alternative to phosphatidylserine extracted from bovine cortex.

Occurrence of phosphatidyl-D-serine in the rat cerebrum.

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction.

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli.

Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains.

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.

Temperature optimization for improved determination of phosphatidylserine species by micro liquid chromatography with electrospray tandem mass spectrometric detection.

A sensitive method for determination of disaturated phosphatidylserine species in the presence of their monounsaturated analogs has been developed, using micro liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The hydrophobic nature of the phosphatidylserine species required a combination of low-eluting sample solvents and sub-ambient temperatures in order to focus large sample volumes up to 20 microL. The samples were dissolved in 2-propanol:hexane:water (20:10:4, v/v/v) prior to 1:9 dilution with ammonium formate buffer:2-propanol:tetrahydrofuran (30:55:15, v/v/v) and final 1:4 dilution with ammonium formate buffer (10 mM):2-propanol: tetrahydrofuran (55:37.5:7.5, v/v/v). The analytical column was a 0.5 x 150 mm stainless steel column packed with 5 microm C30 particles, while the mobile phase contained ammonium formate buffer (10 mM): 2-propanol:tetrahydrofuran (30:55:15, v/v/v). A temperature program from 5 degrees C (hold for 3 minutes) to 75 degrees C at 8 K/min provided separation of the disaturated phosphatidylserine species from their monounsaturated analogs, making available a sensitive determination of the isobaric species. The mass limit of detection for dipalmitoyl phosphatidylserine was 100 pg, corresponding to a concentration limit of detection of 5 pg/microL when using an injection volume of 20 microL. This is an improvement by a factor of 20 as compared to previously reported numbers obtained with conventional LC columns. The within-assay precision of dipalmitoyl phosphatidylserine was 11.9% RSD (n = 3), while the retention time precision was 4.1% RSD (n = 6).

Separation and identification of phosphatidylserine molecular species using reversed-phase high-performance liquid chromatography with evaporative light scattering and mass spectrometric detection.

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C(18) and C(30) columns. The best chromatographic resolution was achieved with the C(30) column. The limit of detection for DPPS was 5 microg/ml (S/N=3) with ELS detection and 0.1 microg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.

Phase separation of cholesterol and the interaction of ethanol with phosphatidylserine-cholesterol bilayer membranes.

Thermotropic and structural effects of ethanol on phosphatidylserine (PS) membranes containing up to 0.4 mol fraction cholesterol were investigated by differential scanning calorimetry, X-ray diffraction and fluorescence spectroscopy. It was found that in the presence of cholesterol, 10% (v/v) added ethanol depresses the melting temperature of the phospholipid by approximately 2 degrees C, similar to what was observed in the absence of cholesterol. Below the melting temperature the progressive disordering effect of added cholesterol is weakly enhanced by the presence of ethanol. In the liquid crystalline state, the marked decrease in the thickness of the bilayer which ethanol causes in the absence of cholesterol (Chem. Phys. Lipids 92 (1998) 127), is also observed in its presence. We conclude that, in contrast to what has been observed for zwitterionic phospholipids, high concentrations of cholesterol do not diminish the interaction of ethanol with PS membranes. With addition of 10% (v/v) ethanol, crystalline cholesterol diffraction, an indication of phase separation of the sterol, appears at mol fraction cholesterol 0.34, as compared to 0.3 in the absence of ethanol (Chem. Phys. Lipids 92 (1998) 71).

A method to study apoptosis in eosinophils by flow cytometry.

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

Brownian ratchets: molecular separations in lipid bilayers supported on patterned arrays.

Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.

Separation procedures for phosphatidylserines.

This paper reviews working procedures for the separation of phosphatidylserines (PS) in complex sample matrices. It begins with an introductory overview of important aspects of PS involvement in cellular lipid biochemistry. The main body of the review describes various procedures for the extraction, isolation, purification, and separation of the PS class and its molecular species in tissue samples. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques. Factors influencing normal-phase and reversed-phase separations are delineated. The last section covers selected chemical derivatization procedures useful for enhancing the separation efficiency and detection sensitivity and specificity.