Marine Algae Membrane Phospholipids Study by High Resolution 31P NMR

Abstract

Membrane phospholipids were extracted using a modified Folch, Lees and Sloane-Stanley method, from 21 different algae species covering three major divisions of the protista kingdom. In the modified method after chloroform/methanol (2:1 v/v) extraction and filtration, the solution was backwashed with K-EDTA, 0.6 M, instead of KCl, 1 M. Because algae samples are eavily loaded with cations that broaden NMR signals, the K-EDTA wash results in more highly resolved NMR signals. Following rotary evaporation, the crude algae lipid extract was dissolved in the chloroform-benzene(d6)/methanol-CsEDTA (2:l ml/ml) reagent and analyzed using a 500 MHz NMR spectrophotometer. Phospholipid chemical shifts were determined relative to standard phosphoric acid (85%), following the UIPAC convention. The internal reference used was phosphatidylcholine (PC, -0.84 δ) Division chlorophyta (8 sps.) yields phospholipid signals for phosphatidylglycerol (PG, 0.50), phosphatidic acid (PA, 0.25), cardiolipin (CL, 0.18), phosphatidylethanolamine (PE, 0.03), sphyngomyelin (SPH, -0.09), phosphatidylinositol (PI, -0.37) and PC; the lysoderivatives for lyso PG (LPG, 1.09) and lyso PC (LPC, -0.28), and one uncharacterized signal at 0.32. Phosphatidylserine (PS -0.05) and plasmalogens were not detected. Division rhodophyta (10 sps.) shows signal from PG, PA, CL, PE, SPH, PI, and PC; the lysoderivatives of lyso PA (LPA, 0.83), lyso PE (LPE, 0.43) and LPC; the plasmalogens PC plasmalogen (PC plas, -0.77), LPC plas (-0.20), and l-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF-acether, -0.70); and an uncharacterized signal at -40 δ chemical shift. PS was not detected. Division Phaeophytas (3 sps.) showed signals for PG, PA, CL, PE, SPH, PI, and PC and lysoderivatives of LPG, LPA, LPE plas (0.53), LPE, LPC plas, and LPC. PS, PAF-acether and the uncharacterized signals at 0.32 δ and -0.40 δ were not detected.     

Phospholipids Analysis by 31P NMR

Abstract

Cell membrane phospholipids can be identified and quantitated using ³¹P NMR spectroscopy in conjunction with an analytical reagent composed of chloroform-benzene(d6)/methanol-CsEDTA 2:l ml/ml. 3 ml of this reagent dissolves between 0.01–100 mg crude tissue lipids obtained by the Folch procedure. When the source phospholipids are strongly contaminated with cations, it is necessary to modify the extraction method, backwashing with K-EDTA, 0.6 M, pH 6, instead of KC1. Also if source tissues must be stored for long periods of time, acetone desication is recommended. Using a 500 MHz ³¹PNMR spectrophotometer (magnetic field ?11.75 T), the extracted phospholipids yield narrow Lorenzian signals (1.8–3.2 Hz at half-height), with these widths at half-height corresponding to their 1/πT₂ values. Chemical shifts (δ) at 24 °C, following the IUPAC shift convention and relative to 85% phosphoric acid, were determined as follows:CAEP,21.09;LPG,l,O9;LPA,0.83;LPE plas,0.53;PG,0.50;LPE,0.43; PA,0.25;CL,0.18;LPI,0.10;PE plas,0.07;PE,0.03;PS,?0.O5;SPH,?0.O9; DiMePE,?b.18;LPC plas,?0.20;LPC,?0.28;PI,?0.37; PAF, ?0.70;PC plas,?0.77; PC, ?0.84. This reagent permits assays of high precision and accuracy that use little spectrometer time and that are suitable for automated procedures.     

Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: the determination of choline containing compounds in foods

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method using multiple scan modes was developed to separate and quantify 11 compounds and lipid classes including acetylcholine (AcCho), betaine (Bet), choline (Cho), glycerophosphocholine (GPC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphocholine (PCho) and sphingomyelin (SM). This includes all of the major choline-containing compounds found in foods. The method offers advantages over other LC methods since HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The LC-MS/MS method allows quantification of all choline-containing compounds in a single run. Tests of method suitability indicated linear ranges of approximately 0.25-25 μg/ml for PI and PE, 0.5-50 μg/ml for PC, 0.05-5 μg/ml for SM and LPC, 0.5-25 μg/ml for LPE, 0.02-5 μg/ml for Cho, and 0.08-8 μg/ml for Bet, respectively. Accuracies of 83-105% with precisions of 1.6-13.2% RSD were achieved for standards over a wide range of concentrations, demonstrating that this method will be suitable for food analysis. 8 polar lipid classes were found in a lipid extract of egg yolk and different species of the same class were differentiated based on their molecular weights and fragment ion information. PC and PE were found to be the most abundant lipid classes consisting of 71% and 18% of the total phospholipids in egg yolk.     

Phospholipid Profiles of Neoplastic Human Breast Tissues

Abstract

Membrane phospholipids from malignant, benign and non-involved human breast tissues were extracted by chloroform-methanol (2:l) and analyzed by ³¹P MR spectroscopy at 202.4 MHz. Fourteen phospholipids were identified as constituents of the profiles obtained among the 52 specimens of the three groups: PC, PC plas LPC, LPC plas, PE, PE plas, LPE, PS, SPH, PI, CL, PG, PA and one uncharacterized resonance at 0.13 6. The relative P-lipid profile mole percentages of phosphorus and indices representing sums and ratios of individual or grouped P-lipids were computed and analyzed by one-way analysis of variance and were compared as simple and complex statistical contrasts. The analysis permitted differentiation among the three groups with the most poignant simple contrast being the relative absence of PA in normal tissues, followed by the significant mean mole percentage differences in PC plas between noninvolved (3.09 ± 0.41) and malignant (4.49 ± 0.23) tissues and between these same tissues in the value, of the PC plas/PC index with mean values of 0.07 ± 0.01 and 0.10 ± 0.006, respectively. Significant complex contrasts were seen between the combined neoplastic tissues and noninvolved tissue in PE plas, LPC plas, PC plas and the (PA/Total Phosphatides-PA) index. Differences were also seen between malignant and non-malignant tissues in LPC, the LPC/PC, LPE/PE and PC plas/PC indices. Differentiation among histologically classified human breast tissues is possible with phospholipid profile analysis and metabolic insight.     

Simultaneous Quantitative Analysis for Phospholipids in Human HDL and LDL Using Two Internal Standards by Liquid Chromatography/Mass Spectrometry

Abstract

A liquid chromatography/mass spectrometry method using two internal standards was developed for the simultaneous quantitative determination of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (LPC) in human high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL). We evaluated this method by examining precision, accuracy, and recovery of phospholipid concentrations in several matrixes. We obtained the time course of phospholipid content in human HDL and LDL treated with sPLA₂‐X and quantitatively observed the decrease of PC, PI, PE, and the increase of LPC. This method should be useful for examination of simultaneous change of endogenous phospholipids in various enzymatic assays.     

A clean‐up technology for the simultaneous determination of lysophosphatidic acid and sphingosine‐1‐phosphate by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry using a phosphate‐capture molecule,Phos‐tag

Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are growth factor‐like lipids having a phosphate group. The concentrations of these mediator lipids in blood are considered to be potential biomarkers for early detection of cancer or vascular diseases. Here, we report a method for simultaneous determination of LPA and S1P using Phos‐tag, a zinc complex that specifically binds to a phosphate‐monoester group. Although both LPA and S1P are hydrophilic compounds, we found that they acquire hydrophobic properties when they form complexes with Phos‐tag. Based on this finding, we developed a method for the enrichment of LPA and S1P from biological samples. The first partition in a two‐phase solvent system consisting of chloroform/methanol/water (1:1:0.9, v/v/v) is conducted for the removal of lipids. LPA and S1P are specifically extracted as Phos‐tag complexes at the second partition by adding Phos‐tag. The Phos‐tag complexes of LPA and S1P are detectable by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) and quantifiable based on the relative intensities of ions using 17:0 LPA and C17 S1P as internal standards. The protocol was validated by analyses of these mediator lipids in calf serum, a rat brain and a lung. The clean‐up protocol is rapid, requires neither thin‐layer chromatography (TLC) nor liquid chromatography (LC), and is applicable to both blood and solid tissue samples. We believe that our protocol will be useful for a routine analysis of LPA and S1P in many clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Phenylmethanesulfonyl fluoride pretreatment stabilizes plasma lipidome in lipidomic and metabolomic analysis

Though it is standard practice to test the stability of analytes in the matrix for routine bioanalytical method, stability evaluation is always impractical and skipped in untargeted lipidomic and metabolomic analysis because analytes in these studies are enormous, diverse and sometimes unknown. Lipidome represents a major class of plasma metabolome and shows great potential to be diagnostic and prognostic biomarkers. However, lipidome also faces stability problems because plasma contains kinds of lipid degradation enzyme. Here, using liquid chromatography time of flight mass spectrometry based lipidomic methodology, plasma levels of various lipids including triglyceride (TG), diglyceride (DG), free fatty acid (FFA), phosphatidylethanolamine (PE) phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), and sphingomyelin (SM) were dynamically determined within 4 h at ambient temperature. In mouse and rat plasma, the levels of most TG, DG, PC and PE species significantly decreased with respect to time, whereas those of LPC, LPE and FFA significantly increased with respect to time. However, such changes did not occur in human plasma, thus indicating hepatic lipase and esterase might involve in the species-specified degradation of lipid classes in plasma. Phenylmethanesulfonyl fluoride (PMSF) pretreatment prevented such lipidome instability in mouse plasma. The results suggested the instability of plasma lipidome should be highly concerned, and the enhancement of ex vivo stability of plasma lipidome could enable more reliable clinical translation of lipidomic data for biomarker discovery.     

Visualization of phosphatidylcholine,lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry

The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.     

Profiling of lipids in Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry

There is evidence from our current research on resistance to stibigluconate and from some previous observations that lipid composition may be altered in resistant Leishmania donovani and in order to explore this we required a comprehensive lipidomics method. Phospholipids can be analysed by direct infusion into a mass spectrometer and such methods can work very well. However, chromatographic methods can also be very effective and are extensively used. They potentially avoid ion suppression effects, associate lipid classes with a retention time range and deliver good quantitative accuracy. In the current study three chromatography columns were compared for their ability to separate different classes of lipid. Butylsilane (C‐4), Zic‐HILIC and a silica gel column were compared. The best results were obtained with a silica gel column used in hydrophilic interaction chromatography (HILIC) mode with a mobile phase gradient consisting of (A) 20% isopropyl alcohol (IPA) in acetonitrile (v/v) and (B) 20% IPA in 0.02 M ammonium formate. Using these conditions separate peaks were obtained for triglycerides (TG), phosphoinositols (PI), inositol phosphoceramides (IPC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidylcholines (PC), sphingosines (SG), lysophosphatidyethanolamines (LPE) and lysophosphatidylcholines (LPC). The methodology was applied to the analysis of lipid extracts from Leishmania donovani and by coupling the chromatography with an LTQ Orbitrap mass spectrometer. It was possible to detect 188 lipid species in the extracts with the following breakdown: PC 59, PE 38, TG 35, PI 20, CPI 13, LPC 11, LPE 2 and SG 10. The fatty acid composition of the more abundant lipids was characterised by MS² and MS³ experiments carried out by using an LCQ Deca low‐resolution ion trap instrument coupled with the silica gel column. The separation of lipids into well‐defined groups gives extra confidence in their identification and minimises the risk of ion suppression effects. High‐resolution mass spectrometry was necessary in order to be able to differentiate between acyl‐ and acyl‐alkyl‐lipids. Copyright © 2010 John Wiley & Sons, Ltd.     

Tissue imaging and serum lipidomic profiling for screening potential biomarkers of thyroid tumors by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

Changes in serum lipidome and in tissue lipidome are associated with cancer. In this study, tissue mass spectrometry imaging (MSI) and serum lipid profiling by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) were performed to investigate significantly changed lipids in both tumor (malignant thyroid cancer (MTC) and benign thyroid tumor (BTT)) tissues and sera. Y-scatterplots of variable importance in the projection (VIP) values vs. fold change values indicate that change trends in the levels of ten lipids (i.e., phosphatidylcholine (PC)(34:1), PC(36:1), PC(38:6), phosphatidic acid (PA) (36:2), PA(36:3), PA(38:3), PA(38:4), PA(38:5), PA(40:5), and sphingomyelin (SM)(34:1)) in both tissues and sera from MTC patients, BTT patients, and normal individuals are significantly associated with these three types of pathophysiological status. In order to examine their diagnostic ability, 289 serum samples from 124 MTC patients, 43 BTT patients, and 122 normal controls were randomly divided into the training set and validation set. A biomarker of PC(34:1) exhibited excellent diagnostic ability to differentiate both MTC and BTT patients from normal individuals, with an area under the receiver operating characteristic (ROC) curve value of 0.984, a sensitivity of 96.4 %, and a specificity of 92.7 %. A panel which included PA(36:3) and SM(34:1) could distinguish between MTC and BTT, with an area under receiver operating characteristic curve (AUC) of 0.961, a sensitivity of 87.8 %, and a specificity of 92.9 %. It is worth noting that a panel consisting of PC(34:1), PA(36:3), and SM(34:1) could differentiate MTC patients from both BTT patients and normal individuals, with an AUC of 0.841, a sensitivity of 86.6 %, and a specificity of 75.5 %.

A single amino acid determines preference between phospholipids and reveals length restriction for activation ofthe S1P<Subscript>4</Subscript> receptor

Background

Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P₁ from S1P to 18:1 LPA.

Results

We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P₄. This mutation severely affected the response of S1P₄ to S1P in a [³⁵S]GTPγS binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P₄ receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors.

Conclusions

Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P₁ receptor.

     

Identification of glycerophospholipid fatty acid remodeling by using mass spectrometry imaging in bisphenol S induced mouse liver

Exposure to BPS induced glycerophospholipid fatty acid remodeling, which might be useful in toxicity evaluation for bisphenols-induced hepatic diseases.     

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.     

Effects of albumin and erythrocyte membranes on spread monolayers of lung surfactant lipids

Dipalmitoyl phosphatidylcholine (DPPC), one of the main constituents of lung surfactant is mainly responsible for reduction of surface tension to near 0 mN/m during expiration, resisting alveolar collapse. Other unsaturated phospholipids like palmitoyloleoyl phosphatidylglycerol (PG), palmitoyloleoyl phosphatidylcholine (POPC) and neutral lipids help in adsorption of lung surfactant to the air-aqueous interface. Lung surfactant lipids may interact with plasma proteins and hematological agents flooding the alveoli in diseased states. In this study, we evaluated the effects of albumin and erythrocyte membranes on spread films of DPPC alone and mixtures of DPPC with each of PG, POPC, palmitoyloleoyl phosphatidylethanolamine (PE), cholesterol (CHOL) and palmitic acid (PA) in 9:1 molar ratios. Surface tension-area isotherms were recorded using a Langmuir-Blodgett (LB) trough at 37 degrees C with 0.9% saline as the sub-phase. In the presence of erythrocyte membranes, DPPC and DPPC+PA monolayers reached minimum surface tensions of 7.3+/-0.9 and 9.6+/-1.4 mN/m, respectively. Other lipid combinations reached significantly higher minimum surface tensions >18 mN/m in presence of membranes (Newman Keul's test, p<0.05). The relative susceptibility to membrane inhibition was [(DPPC+PG, 7:3)=(DPPC+PG, 9:1)=(DPPC+POPC)=(DPPC+PE)=(DPPC+CHOL)]>[(DPPC+PA)=(DPPC)]. The differential response was more pronounced in case of albumin with DPPC and DPPC+PA monolayers reaching minimum surface tensions less than 2.4 mN/m in presence of albumin, whereas DPPC+PG and DPPC+POPC reached minimum surface tensions of around 20 mN/m in presence of albumin. Descending order of susceptibility of the spread monolayers of lipid mixtures to albumin destabilization was as follows: [(DPPC+PG, 7:3)=(DPPC+PG, 9:1)=(DPPC+POPC)]>[(DPPC+PE)=(DPPC+CHOL)]>[(DPPC+PA)=(DPPC)] The increase in minimum surface tension in presence of albumin and erythrocyte membranes was accompanied by sudden increases in compressibility at surface tensions of 15-30 mN/m. This suggests a monolayer destabilization and could be indicative of phase transitions in the mixed lipid films due to the presence of the hydrophobic constituents of erythrocyte membranes.     

High performance liquid chromatography-tandem mass spectrometry of phospholipid molecular species in eggs from hens fed diets enriched in seal blubber oil

The total lipid fraction of eggs from hens fed diets enriched in seal blubber oil (1.25-5.0% SBO) was directly analysed with normal-phase high performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC-ESI-MS-MS) for the identification of the molecular species of phospholipids (PLs). The species of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were all detected as the [M-H](-) ions. The phosphatidylcholine (PC), sphingomyelin (Sph) and lysophosphatidylcholine (LPC) classes, were detected as formate adducts [M+HCOO](-). Tandem MS of PE and PI showed the loss of the carboxylate anions, and, for PI, also the loss of water and inositol. Product ion spectrum of PC, LPC and Sph contained only the [M-CH(3)](-) ion fragment. Feeding different levels of SBO for 5 weeks resulted in a significant increase of PE, PC and PI molecular species carrying eicosapentaenoic acid (C(20:5 omega3), EPA), docosapentaenoic acid (C(22:5 omega3), DPA) and docosahexaenoic acid (C(22:6 omega3), DHA), but not Sph nor LPC. The highest increase of the omega3/omega6 ratio occurred for PE and PC. On the contrary, PI was less affected by the increase of SBO in the diet.     

Action of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis is significantly influenced by coexisting lipids in substrate-detergent micelles.

The effects of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol on the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail in phosphatidylinositol (PI)/detergent mixed micelles. By addition of PC, the enzymatic hydrolysis of PI was significantly stimulated in PI/Triton X-100 as well as PI/sodium deoxycholate (SDC) mixed micelles. SM stimulated enzyme activity toward PI/Triton X-100 micelles at a lower molar ratio of SM to PI, but was rather inhibitory at a ratio higher than 2.0. The enzyme activity became significantly lower with an increase of PE or cholesterol in PI/Triton X-100 micelles. Actually, both PE and cholesterol were intensively inhibitory when added at a higher molar ratio to PI in Triton X-100-containing micelles. In the system of PI/SDC mixed micelles, not only PC but also SM, PE and cholesterol enhanced the enzymatic hydrolysis of PI. The difference between PI/Triton X-100 and PI/SDC micelles regarding the effects of these lipids on PI-PLC action, must be dependent on the physical state of micelles formed by these detergents and lipids.     

Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids

In this work we report the development of a novel methodology for the determination of stereospecificity of diacyl glycerophospholipids, including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols (PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized in negative ion mode. This methodology uses MS(2) recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2](-) ) and as ketenes ([M-(Sn2-H(2) O)](-) ) exhibited consistently higher intensity than their counterpart ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1](-) and [M-(Sn1-H(2) O)](-) ). Therefore, we concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions. We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined. Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans (MPIS), fatty acyl scans (FAS), and multidimensional mass spectrometry based shotgun lipidomics (MDMS-SL), should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling.     

Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer's disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC-MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole-time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.     

Application of supercritical fluid chromatography to the analysis of hydrophobic metabolites

This review describes the usefulness of supercritical fluid chromatography (SFC) in the analysis of hydrophobic metabolites. The use of SFC for the analysis of naturally occurring polyprenols markedly improves the chromatographic resolution of polyprenol homologues and their geometric isomers as compared to conventional HPLC. Under optimized SFC conditions, individual homologues with 10-100-mers were separated. Furthermore, we established an analytical system for the fingerprinting and profiling of diverse lipids through the usage of SFC-MS. When a cyanopropylated silica gel packed column was used for the separation, 14 lipids were successfully detected, and the time required for analysis was less than 15 min. The use of an octadecylsilylated column for the separation depended on the differences in the fatty acid side chains. SFC is a useful separation technology for hydrophobic metabolites, which are difficult to be separated by HPLC.