Liquid chromatography with dual parallel mass spectrometry and (31)P nuclear magnetic resonance spectroscopy for analysis of sphingomyelin and dihydrosphingomyelin. I. Bovine brain and chicken egg yolk

Liquid chromatography coupled to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) mass spectrometry (MS), in parallel, was used for detection of bovine brain and chicken egg sphingolipids (SLs). APCI-MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H(2)O+H](+) and [Cer-2H(2)O+H](+), whereas ESI-MS produced mostly intact protonated molecules, [M+H](+). APCI-MS/MS and MS(3) were used to differentiate between isobaric SLs. APCI-MS/MS mass spectra exhibited long-chain base related fragments, [LCB](+) and [LCB-H(2)O](+), that allowed the sphinganine backbone to be differentiated from the sphingenine backbone. Fragments formed from the fatty amide chain, [FA(long)](+) and [FA(short)](+), allowed an overall fatty acid composition to be determined. The presence of both dihydrosphingomyelin (DSM) and sphingomyelin (SM) sphingolipid classes was confirmed using (31)P NMR spectroscopy.     

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.     

Studies on sulfatides by quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes that include an unusual internal galactose residue loss and the classical charge-remote fragmentation

The structural characterization of sulfatides by collisional-activated dissociation (CAD) quadrupole ion-trap tandem mass spectrometric methods with electrospray ionization is described. When subjected to CAD in the negative-ion mode, the [M - H]- ions of sulfatides yield abundant structurally informative ions that permit unequivocal assignments of the long-chain base, and fatty acid constituent including the location of double bond. The identification of the position of the double bond on the fatty acyl substituent is based on the observation of the series of the ions arising from classical charge-remote fragmentation processes similar to those observed by high-energy CAD and by tandem quadrupole mass spectrometry. An unusual internal galactose residue loss due to a rearrangement process was also observed. The [M - H]- ions of sulfatides also dissociates to a ceramide anion, which undergoes consecutive fragmentation processes to yield ions informative for identification of the ceramide moiety and permits distinction the sulfatide with a sphingosine subclass from that with a sphinganine long-chain base subclass. The MS(2)-spectra of the sulfatide subclass with a sphingosine LCB and a alpha-hydroxy fatty acyl substituent (d18:1/hFA-sulfatide) are featured by the prominent ion sets of m/z 568, 550, 540, and 522, originated from a primary cleavage of the fatty acyl CO-CH(OH) bond, and are readily differentiable from those arising from the non-hydroxy sulfatide subclass (d18:1/nFA-sulfatide), in which the ion sets are of low abundance. The fragmentation pathways of sulfatides under low-energy CAD are proposed. The pathways are supported by the MS(2)- and MS(3)-spectra of various compounds, and of their H-D exchanged analogs.     

High-resolution analysis by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for the identification of molecular species of phospholipids and their oxidized metabolites

Nano-electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was applied to identify the molecular species of phosphatidylethanolamine of Caenorhabditis elegans, which has a high concentration of phospholipids with a fatty acyl chain having an odd number of carbon atoms. The molecular species of diacyl phosphatidylethanolamine with one fatty acyl chain having an odd number of carbon atoms and one fatty acyl chain having an even number of carbon atoms was identified separately from alkyl-acyl phosphatidylethanolamine with an alkyl chain having an even number of carbon atoms and a fatty acyl chain having an even number of carbon atoms. Furthermore, nano-ESI-FTICRMS was applied to the direct identification of oxidized phosphatidylcholine from soybean. The mass peaks of individual molecular species of oxidative phosphatidylcholine, such as 34:3 diacyl phosphatidylcholine with peroxide (+2O) (m/z 788.544) or 34:2 diacyl phosphatidylcholine with peroxide (+2O) (m/z 790.560), can be separated from the peaks of the molecular species of the non-oxidative phospholipids. This suggests that the mass peaks with a difference of less than 0.1 mass units in their molecular weight can be separated and that their individual exact molecular compositions can be obtained by the FTICRMS analysis. The high resolution and high accuracy of FTICRMS are very effective in the analysis of molecular species of phospholipids and their derivatives.     

Determination of phosphatidylethanolamine molecular species in various food matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS2)

A liquid chromatographic–electrospray ionization–tandem mass spectrometric (LC–ESI–MS²) method has been developed for determination of the molecular species of phosphatidylethanolamine (PE) in four food matrices (soy, egg yolk, ox liver, and krill oil). The extraction and purification method consisted of a pressurized liquid extraction procedure for total lipid (TL) extraction, purification of phospholipids (PLs) by adsorption on a silica gel column, and separation of PL classes by semi-preparative normal-phase HPLC. Separation and identification of PE molecular species were performed by reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry (ESI–MS²). Methanol containing 5 mmol L⁻¹ ammonium formate was used as the mobile phase. A variety of PE molecular species were detected in the four food matrices. (C16:0–C18:2)PE, (C18:2–C18:2)PE, and (C16:0–C18:1)PE were the major PE molecular species in soy. Egg yolk PE contained (C16:0–C18:1)PE, (C18:0–C18:1)PE, (C18:0–C18:2)PE, and (C16:0–C18:2)PE as the major molecular species. Ox liver PE was rich in the species (C18:0–C18:1)PE, (C18:0–C20:4)PE, and (C18:0–C18:2)PE. Finally, krill oil which was particularly rich in (C16:0(alkyl)–C22:6(acyl))plasmanylethanolamine (PakE), (C16:0–C22:6)PE, and (C16:0–C20:5)PE, seemed to be an interesting potential source for supplementation of food with eicosapentaenoic acid and docosahexaenoic acid.     

Molecular dynamics simulations of bilayers containing mixtures of sphingomyelin with cholesterol and phosphatidylcholine with cholesterol

We performed six molecular dynamics simulations: three on hydrated bilayers containing pure phospholipids and three on hydrated bilayers containing mixtures of these phospholipids with cholesterol. The phospholipids in our simulations were SSM (sphingomyelin containing a saturated 18:0 acyl chain), OSM (sphingomyelin with an unsaturated 18:1 acyl chain), and POPC (palmitoyloleoylphosphatidylcholine containing one saturated and one unsaturated chain). Data from our simulations were used to study systematically the effect of cholesterol on phospholipids that differed in their headgroup and tail composition. In addition to the structural analysis, we performed an energetic analysis and observed that energies of interaction between cholesterol and neighboring SM molecules are similar to the energies of interaction between cholesterol and POPC. We also observed that the interaction energy between cholesterol and neighboring lipids cannot be used for the determination of which lipids are involved in the creation of a complex.     

Characterization of inositol phosphorylceramides from <Emphasis Type="Italic">Leishmania major</Emphasis> by tandem mass spectrometry with electrospray ionization

We describe tandem mass spectrometric approaches, including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species seen as [M - H](-) and [M - 2H + Li](-) ions in the negative-ion mode as well as [M + H](+), [M + Li](+), and [M - H + 2Li](+) ions in the positive-ion mode. Following CAD in an ion-trap or a triple-stage quadrupole instrument, the [M - H](-) ions of IPC yielded fragment ions reflecting only the inositol and the fatty acyl substituent of the molecule. In contrast, the mass spectra from MS(3) of [M - H - Inositol](-) ions contained abundant ions that are readily applicable for assignment of the fatty acid and long-chain base (LCB) moieties. Both the product-ion spectra from MS(2) and MS(3) of the [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) ions also contained rich fragment ions informative for unambiguous assignment of the fatty acyl substituent and the LCB. However, the sensitivity of the ions observed in the forms of [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) (Alk = Li, Na) is nearly 10 times less than that observed in the [M - H](-) form. In addition to the major fragmentation pathways leading to elimination of the inositol or inositol monophosphate moiety, several structurally informative ions resulting from rearrangement processes were observed. The fragmentation processes are similar to those previously reported for ceramides. While the tandem mass spectrometric approach using MS(n) (n = 2, 3) permits the structures of the Leishmania major IPCs consisting of two isomeric structures to be unveiled in detail, tandem mass spectra from constant neutral loss scans may provide a simple method for detecting IPC in mixtures.     

Electron capture dissociation of complexes of diacylglycerophosphocholine and divalent metal ions: Competition between charge reduction and radical induced phospholipid fragmentation

Divalent metal complexes of phosphocholines, [Metal(II)(L)(n)](2+) (where Metal=Cu(2+), Co(2+), Mg(2+), and Ca(2+), L=1,2-dihexanoyl-sn-glycero-3-phosphocholine [6:0/6:0GPCho] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [16:0/18:1GPCho] and n=2-5), were formed upon electrospray ionization mass spectrometry (ESI/MS) of 8 mM solution of phosphocholine (L) with 4 mM metal salt (Metal). The electron capture dissociation (ECD) reactions of these [Metal(II)(L)(n)](2+) complexes were examined via Fourier-transform ion-cyclotron resonance mass spectrometry. A rich and complex chemistry was observed, including charge reduction and fragmentation involving losses of a methyl radical, trimethylamine, and the acyl chains. The predominant reaction channel was dependent on the size (n) of the complex, the metal and ligand used, and the size of the acyl chain. Thus charge reduction dominates the ECD spectra of the larger phosphocholine, 16:0/18:1GPCho, but is largely absent in the smaller 6:0/6:0GPCho. For complexes of 16:0/18:1GPCho, n=4-5, fragmentation from the head group mainly occurs via loss of the methyl radical and trimethylamine. At n=3, the relative abundance of fragments due to loss of acyl chain radicals increases. The abundances of ions arising from these radical losses increase further for the n=2 complexes, thereby providing information on the composition and position of the 16:0 and 18:1 acyl groups. Thus ECD of metal complexes provides structurally useful information on the phosphocholine, including the nature of the head group, the acyl chains, and the positions of the acyl chains.     

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS)

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSⁿ, n = 2–4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M−H]⁻ at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M−H]⁻ ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.     

Lipidomics of the Edible Brown Alga Wakame (Undaria pinnatifida) by Liquid Chromatography Coupled to Electrospray Ionization and Tandem Mass Spectrometry

The lipidome of a brown seaweed commonly known as wakame (Undaria pinnatifida), which is grown and consumed around the world, including Western countries, as a healthy nutraceutical food or supplement, was here extensively examined. The study was focused on the characterization of phospholipids (PL) and glycolipids (GL) by liquid chromatography (LC), either hydrophilic interaction LC (HILIC) or reversed-phase LC (RPLC), coupled to electrospray ionization (ESI) and mass spectrometry (MS), operated both in high and in low-resolution mode. Through the acquisition of single (MS) and tandem (MS/MS) mass spectra more than 200 PL and GL of U. pinnatifida extracts were characterized in terms of lipid class, fatty acyl (FA) chain composition (length and number of unsaturations), and regiochemistry, namely 16 SQDG, 6 SQMG, 12 DGDG, 5 DGMG, 29 PG, 8 LPG, 19 PI, 14 PA, 19 PE, 8 PE, 38 PC, and 27 LPC. The FA (C16:0) was the most abundant saturated acyl chain, whereas the monounsaturated C18:1 and the polyunsaturated C18:2 and C20:4 chains were the prevailing ones. Odd-numbered acyl chains, iJ., C15:0, C17:0, C19:0, and C19:1, were also recognized. While SQDG exhibited the longest and most unsaturated acyl chains, C18:1, C18:2, and C18:3, in the sn-1 position of glycerol, they were preferentially located in the sn-2 position in the case of PL. The developed analytical approach might pave the way to extend lipidomic investigations also for other edible marine algae, thus emphasizing their potential role as a source of bioactive lipids.     

Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation pathways of ceramides under low energy collisional-activated dissociation (CAD) by studying more than thirty compounds in nine subclasses. The product-ion spectra of the [M + Li]+ ions of ceramides contain abundant fragment ions that identify the fatty acyl substituent and the long-chain base (LCB) of the molecules, and thus, the structure of ceramides can be easily determined. Fragment ions specific to each ceramide subclasses are also observed. These feature ions permit differentiation among different ceramide subclasses. The ion series arising from the classical C-C bond cleavages that were reported in the fast-atom bombardment (FAB)-high energy tandem mass spectrometry is not observable; however, the product-ion spectra contain multiple fragment ions informative for structural characterization and isomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source CAD (pseudo-MS3) and of the deuterium-labeling molecular species obtained by H/D exchange to support the ion structure assignments and the proposed fragmentation pathways that lead to the ion formation.     

Electrospray ionization multiple stage quadrupole ion-trap and tandem quadrupole mass spectrometric studies on phosphatidylglycerol from arabidopsis leaves

Phosphatidylglycerol (PG) is the major phospholipid of plant chloroplasts. PG from Arabidopsis thaliana has an unusual fatty acyl chain, 3-trans-hexadecenoyl (Delta(3)16:1) in the sn-2 position of the major 18:3/Delta(3)16:1-PG species, as well as in 18:2/Delta(3)16:1-PG and 16:0/Delta(3)16:1-PG. Upon low-energy collisionally activated dissociation (CAD) in a tandem quadrupole or in an ion-trap mass spectrometer, the [M - H]- ions of the PG molecules containing Delta(3)16:1 give product-ion spectra that are readily distinguishable from those arising from PGs without the Delta(3)16:1 species. The Delta(3)16:1-fatty acyl-containing PGs are characterized by MS(2) product-ion mass spectra that contain predominant [M - H - 236]- ions arising from loss of the Delta(3)16:1-fatty acyl substituent as a ketene. This is attributable to the fact that the alpha-hydrogen of the Delta(3)16:1-fatty acid substituent involved in the ketene loss is an allylic hydrogen, which is very labile. This leads to preferential neutral loss of 236 and drastic decline in the neutral loss of 254 (i.e., loss as a fatty acid), the unique features that signify the presence of Delta(3)16:1-fatty acyl containing PGs. The neutral loss scan of 236, thus, provides a sensitive tandem quadrupole mass spectrometric means to identify Delta(3)16:1-containing PG species in lipid mixtures. This low-energy tandem mass spectrometric approach also permits the structures of the Arabidopsis PGs that consist of two isomeric structures to be unveiled.     

Structural property and function of D-erythro asymmetric chain sphingomyelins as studied by microcalorimetry and electron microscopy

D-erythro sphingomyelines (SM) having a defined acyl chain were synthesized with sphingosylphosphorylcholine as a starting material, and both a structural property and its relating phase transition phenomenon were compared between a symmetric chain length SM (palmitoyl-SM: C16-SM) and asymmetric chain length SMs (behenoyl-SM: C22-SM, lignoceryl-SM: C24-SM). Furthermore, effect of increasing a content of asymmetric chain SMs in the mixture systems of C22-SM/C16-SM, and C24-SM/C16-SM was investigated. The present calorimetric and electron microscopic studies revealed that (1) The main transition enthalpy is smaller for the asymmetric chain SMs than for the symmetric chain SM by about 3 kJ mol⁻¹, although the acyl chain length is longer for the former than for latter; (2) Relatively small size vesicles (100∼200 nm diameters) surrounded by one or more lamellae are observed for the asymmetric chain SMs, in contrast to large multilamellar vesicles (1500∼2500 nm diameters) having at least fifteen stained lamellae for the symmetric chain SM and (3) The coexisting asymmetric chain SMs cause the decrease in size and multiplicity for the MLV of the symmetric chain SM, simultaneously with a decrease in the main transition enthalpy.     

Characterization of protonated phospholipids as fragile ions in quadrupole ion trap mass spectrometry

Some ions exhibit "ion fragility" in quadrupole ion trap mass spectrometry (QIT-MS) during mass analysis with resonance ejection. In many cases, different ions generated from the same compound exhibit different degrees of ion fragility, with some ions (e.g., the [M+H](+) ion) stable and other ions (e.g., the [M+Na](+) ion) fragile. The ion fragility for quadrupole ion trap (QIT) mass spectrometry (MS) for protonated and sodiated ions of three phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, PC (16:0/16:0), 1,2-dipalmitoyl-sn-glycero-3-phophoethanolamine, PE (16:0/16:0), and N-palmitoyl-D-erythro-sphingosylphosphorylcholine, SM (d18:1/16:0), was determined using three previously developed experiments: 1) the peak width using a slow scan speed, 2) the width of the isolation window for efficient isolation, and 3) the energy required for collision-induced dissociation. In addition, ion fragility studies were designed and performed to explore a correlation between ion fragility in QIT mass analysis and ion fragility during transport between the ion source and the ion trap. These experiments were: 1) evaluating the amount of thermal-induced dissociation as a function of heated capillary temperature, and 2) determining the extent of fragmentation occurring with increasing tube lens voltage. All phospholipid species studied exhibited greater ion fragility as protonated species in ion trap mass analysis than as sodiated species. In addition, the protonated species of both SM (d18:0/16:0) and PC (16:0/16:0) exhibited greater tendencies to fragment at higher heated capillary temperatures and high tube lens voltages, whereas the PE (16:0/16:0) ions did not appear to exhibit fragility during ion transport.     

Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.     

MALDI Mass Spectrometric Imaging of Lipids in Rat Brain Injury Models

Matrix-assisted laser desorption ionization/ionization imaging mass spectrometry (MALDI IMS) with a time-of-flight analyzer was used to characterize the distribution of lipid molecular species in the brain of rats in two injury models. Ischemia/reperfusion injury of the rat brain after bilateral occlusion of the carotid artery altered appearance of the phospholipids present in the hippocampal region, specifically the CA1 region. These brain regions also had a large increase in the ion abundance at m/z 548.5 and collisional activation supported identification of this ion as arising from ceramide (d18:1/18:0), a lipid known to be associated with cellular apoptosis. Traumatic brain injury model in the rat was examined by MALDI IMS and the area of damage also showed an increase in ceramide (d18:1/18:0) and a remarkable loss of signal for the potassium adduct of the most abundant phosphocholine molecular species 16:0/18:1 (PC) with a corresponding increase in the sodium adduct ion. This change in PC alkali attachment ion was suggested to be a result of edema and influx of extracellular fluid likely through a loss of Na/K-ATPase caused by the injury. These studies reveal the value of MALDI IMS to examine tissues for changes in lipid biochemistry and will provide data needed to eventually understand the biochemical mechanisms relevant to tissue injury.     

Using high-performance liquid chromatography/mass spectrometry for the quantification of plasma phospholipids in children with type 1 diabetes

Quantification of the plasma phospholipids composition in children with different stages of type 1 diabetes was performed using HPLC/MS with electrospray ionization. Reliable (p < 0.05) changes in the concentrations of both the general phospholipid classes and individual molecular species were revealed. It was found that, regardless of the disease duration, the concentration of lysophosphatidylcholine in the plasma was reduced. The concentration of the other phospholipid classes remained virtually unaffected. Quantitative profiling showed that the concentrations of phosphatidylcholine (16: 0/16: 0, 16: 0/18: 1 and 18: 2/20: 4) and phosphatidylethanolamine (16: 0/22: 6) were significantly increased and those of phosphatidylcholine (18: 0/18: 2, 18: 2/18: 2) and sphingomyelin (14: 0/S18: 1, 15: 0/S18: 1, 22: 2/S18: 0, and 24: 2/S18: 0) were reduced in the plasma at the early stages of type 1 diabetes. A similar dynamics was observed for phosphatidylcholine 16: 0/20: 4 molecular species and plasmalogen phosphatidylcholine 16: 0/20: 4, namely, an increase in the concentration of phospholipids containing arachidonic acid at the early stages of the disease and normalization of the prolong disease.     

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.     

MALDI-mass spectrometry imaging of desalted rat brain sections reveals ischemia-mediated changes of lipids

Ischemia-mediated lipidomic changes in rat brains were explored by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) profiling and imaging after in situ desalting which drastically simplified the spectral presentation of tissue lipids. Removal of interference from the massively changed cations in response to tissue damage permitted the revelation of subtle yet important lipidomic changes. The identities of the detected lipids were confirmed by MALDI tandem mass spectrometry (MALDI-MS/MS). The MALDI-MS imaging (MALDI-MSI) result of lysophosphatidylcholine 16:0 (LPC 16:0) in the desalted brain section appeared essentially identical to that of sodiated LPC 16:0 in the adjacent undesalted section and verified the suitability of the desalting method for the MALDI-MSI studies of lipids in tissue. Other than the consistently decreased phosphatidylcholine (PC) 16:0/18:1, images of PCs containing all saturated, or combined saturated and monounsaturated fatty acyl (MUFA) residues revealed their parenchymal increase by ischemia. Images of PCs containing polyunsaturated fatty acyl (PUFA) residues in normal cortex showed laminated patterns similar to cortical lamina. Ischemia reduced the abundance of PC 16:0/20:4 and PC 16:0/22:6 and disrupted the laminated distribution of the former. However, ischemia increased the subcortical abundance of PUFA-PCs containing stearoyl residue and confined their cortical increase within limited areas. Image of parenchymal sphingomyelin 18:0 (SM 18:0) showed its consistent decrease by ischemia that paralleled the increase of ceramide 18:0-H(2)O in region of moderate to high SM abundance. The above results presented the lipidomic changes largely different from previous MALDI-MSI results and suggested a window of intervention that may benefit the management of cerebrovascular accident and other brain injuries.     

液相色谱-四极杆-飞行时间串联质谱法高通量分析美国红参的脑苷脂分子种

利用液相色谱-四极杆-飞行时间串联质谱法(LC-Q-TOF MS/MS)分析美国红参(Parastichopus californicus)脑苷脂的分子种组成。以氯仿-甲醇(2：1,v/v)溶液提取,经皂化和固相萃取柱(SPE)净化后,在正离子模式下,通过自动二级质谱方式同时获取样品脑苷脂分子的一级和二级质谱图。筛取含180 Da中性丢失碎片的分子,再结合二级质谱中的长链碱和脂肪酸碎片,确定每种脑苷脂分子的结构。通过该法一次性共分析出123种美国红参脑苷脂分子种;含有的长链碱共18种,其中植物鞘氨醇型与鞘氨醇型长链碱的相对含量比为1：2;脂肪酸为18~25碳的饱和或单不饱和脂肪酸,以24碳脂肪酸为主,2-羟基脂肪酸占58.62%,饱和与单不饱和脂肪酸的相对含量比约为1：3。LC-Q-TOF MS/MS法分析海参脑苷脂分子种的灵敏度高,准确,简便,为海参脑苷脂的构效关系研究及功能食品开发提供理论依据。