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52nd asms conference abstracts listingAbstracts
Endogenous anti-inflammatory lipid mediators, resolvins and docosatrienes: LC-UV-MS-MS-based lipidomic
analysis, databases, and searching algorithms
Brigham and Women's Hosp./Harvard Medical School, Boston, MA Introduction
Lipidomics holds the promise to decode the identity, structure-function relationships, and information held within
biological systems where lipid domains and lipidic micro- compartments play essential roles in orchestrating complex
physiologic and pathophysiologic events. Mediator-lipidomics is a subsection of metabolomics and lipidomics that
focuses solely on the profiling of bioactive lipid mediators (LM). LM can be grouped into those that act extracellularly
and those that are intracellular mediators. This lecture will review formation and actions of novel mediators of
resolution of acute inflammatory events termed resolvins and protectins. These are examples of extracellular
mediators derived from the omega-3 essential fatty acids. To also illustrate mediator lipidomics, we will present
analysis of diacylglyceride (DAG) that serve as intracellular mediators in signal transduction.
LC-MS-MS methods for lipid mediator lipidomics include LC-tandem UV-MS-MS analyses together with recently
constructed algorithms and databases for essential fatty acid-derived lipid mediators, resolvins, docosatrienes,
protectins, and eicosanoids. In addition, LC-MS-MS analysis of diacylglyceride molecular species was carried out
using LC-MS-MS together with synthetic standards for the major DAG species. Analyses were performed using a
Finnigan LCQ-MAT quadrupole ion-trap mass spectrometer equipped with an electrospray ionization interface.
Specific conditions for the chromatographic and mass spectral analysis of lipid mediators derived from essential fatty
acids and those for the chromatographic analysis and identification of DAG will be presented.
Profiling of LM present within tissues and cells can provide a powerful diagnostic view of complex physiologic and
pathologic events. Recent results obtained using our mediator-lipidomics approach with human diseases are
examples. We shall discuss results obtained with leukocytes from localized aggressive periodontal disease as in “A
molecular defect in intracellular lipid signaling in human neutrophils in localized aggressive periodontal tissue
damage” (J. Immunol. 2004; 172:1856), and extracellular LM analysis of those formed from omega-3 pathways will be
presented (“Stereochemical assignment, anti-inflammatory properties, and receptor for the omega-3 lipid mediator
Resolvin E1,” J. Exp. Med. 2005, in press). These provide examples of the powerful capabilities of lipid mediator
lipidomics when carried out in tandem with in vivo and cellular-based systems.
Lipidomics of cyclooxygenase-mediated oxidative stress
Ian A. Blair; Seon Hwa Lee; Michelle V. Williams; University of Pennsylvania, Philadelphia, PA Introduction
The ability to resolve enantiomeric, regioisomeric, and stereoisomeric bioactive lipids is particularly important for
cyclooxygenase (COX)- and lipoxygenase (LOX)-derived bioactive lipids as well as those arising non-enzymatically
from reactive oxygen species (ROS). From a mechanistic perspective it is important to be able to distinguish these
isomeric compounds with high specificity and sensitivity. It is relatively simple to derivatize bioactive lipids with an
electron-capturing group such as the pentafluorobenzyl (PFB) moiety before LC analysis. In combination with
LC/electron capture APCI/MS it is possible increase sensitivity by two orders of magnitude when compared with
conventional LC/MS methodology and underivatized analytes.
http://www.asms.org/abstracts/DisplayAbstractList.aspx?Session=MODam (1 of 4) [28-May-05 5:32:36 PM] Methods
A Finnigan TSQ 7000 triple stage quadrupole mass spectrometer (Thermo Electron, San Jose) equipped with an
APCI source was used in the studies. For full-scan and MRM analyses, unit resolution was maintained for both parent
and product ions. For the lipidomics profile, the instrument was operated in the negative ion mode. Operating
conditions for the TSQ 7000 were as follows: vaporizer temperature at 500 oC, heated capillary temperature at 230
oC, with the corona discharge needle set at 16 •A. Nitrogen was used for the sheath and auxiliary gas. Collision-
induced dissociation (CID) was performed using argon as the collision gas at 2.7 mTorr in the Rf-only quadrupole.
Targeted chiral LC/electron capture APCI/MRM/MS analysis was conducted using pentafluorobenzyl (PFB)
The targeted lipidomics approach showed that 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] was the major
hydroxylated non-esterified lipid formed in these cells. The corresponding hydperoxide, 15(S)-
hydroperoxyeicosatetraenoic acid [15(S)-HPETE] undergoes homolytic decomposition to the DNA-reactive
bifunctional electrophile 4-oxo-2-nonenal, a precursor of heptanone-etheno-2-deoxyguanosine. This etheno-adduct
was identified in DNA of RIES cells that expressed COX-2. A dose-dependent increase in adduct levels was observed
in the presence of vitamin C. This suggested that vitamin C increased lipid hydroperoxide-mediated 4-oxo-2-nonenal
formation in the cells. The selective cyclooxygenase-2 inhibitor NS-398 was protective against cellular DNA damage
but was less effective if vitamin C was present. Prostaglandin E2 (PGE2) and 15(S)-HETE biosynthesis were
completely inhibited by NS-398. 15(R)-HETE was detected in amounts that were slightly higher than the original 15(S)-
HETE observed in the absence of aspirin, which suggested that significant amounts of 15(R)-HPETE had also been
formed. Thus, the electron capture methodology provided an excellent means to analyze trace amounts of chiral lipids
when chromatography was performed on a chiral column using normal phase solvents. Supported by NIH RO-1
Analysis of Cell Membrane Aminophospholipids as Isotope-Tagged (iTRAQ) Derivatives
Karin A. Zemski Berry; Robert C. Murphy; University of Colorado Health Science Center, Aurora, CO Introduction
Recently a set of four different isotopically enriched N-methylpiperazine acetic acid NHS (N-hydroxysuccinimide) ester
reagents (iTRAQ) has been developed by Applied Biosystems that places isobaric mass labels at any primary amine
group. The resulting derivatized products are isobaric and chromatographically indistinguishable, but yield reporter
ions (m/z 114 or 117) during CID in the positive ion mode that can be used to identify and quantify individual members
of a multiplex set. In this study, phospholipids that contain primary amine groups, such as
glycerophosphatidylethanolamine (GPEtn) and glycerophosphatidylserine (GPSer), were modified using these
reagents and it was established that this modification could aid in the mass spectrometric identification of phospholipid
changes that occur during biological stimuli.
GPSer and GPEtn standards were labeled with 114 and 117 N-methylpiperazine acetic acid NHS ester reagents and
the mass spectrometric, NP-HPLC, and RP-HPLC behavior was investigated using an electrospray triple quadrupole
mass spectrometer. Once the mass spectrometric response of the N-methylpiperazine amide tagged
aminophospholipids was determined, a precursor ion scan of m/z 114 or 117 scan was used to detect these species.
The N-methylpiperazine acetic acid NHS ester reagents were used to assess the changes that occurred in the
distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to
Cu2+/H2O2 in order to determine the feasibility of these reagents to track changes in the distribution of
aminophospholipids after a stimulus.
The mass spectrometric response of N-methylpiperazine amide tagged aminophospholipids was probed using 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine
standards. In the positive ion full scan spectrum the [M+H]+ of each of these N-methylpiperazine amide tagged
aminophospholipids shifted 144 Da and during collision-induced dissociation the major fragmentation ion was at m/z
114 or 117 depending on the specific reagent used. The negative ion CID behavior of N-methylpiperazine amide
tagged GPEtn and GPSer species remained the same compared to non-tagged GPEtn and GPSer species. In order
to determine if these N-methylpiperazine acetic acid NHS ester reagents work well for complex biological samples, the
GPEtn lipids extracted from RAW 264.7 cells were tagged and it was found that the [M+H]+ distribution of GPEtn
species was shifted by 144 Da and that the CID behavior in the positive ion mode of all subclasses of GPEtn is
uniform with a major ion at m/z 114 or 117, which allows all subclasses of tagged GPEtn species to be detected using
a precursors of m/z 114 or 117 scan. Finally, the N-methylpiperazine acetic acid NHS ester reagents were used to
http://www.asms.org/abstracts/DisplayAbstractList.aspx?Session=MODam (2 of 4) [28-May-05 5:32:36 PM] assess the changes that occurred in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2. The control liposomes were labeled with the 114 reagent and the Cu2+/H2O2 liposomes were labeled with the 117 reagent. Upon comparison of the precursors of m/z 114 scan to the precursors of m/z 117 scan it was found that the amount of lyso GPEtn increased in the oxidized sample. It has been found that the N-methylpiperazine amide isotope tag is a novel way to observe changes in the distribution of GPEtn and GPSer species, which has been difficult to achieve in the past. Lipidomics of bacterial invasion and dormancy
Anne K Bendt1; Lorraine D Hernandez2; Karsten Hueffer2; Guanghou Shui1; Wee Kiang Yeo1; Jorge E Galan2; 1National University of Singapore, Singapore, Singapore; 2Yale University , New Haven, CT; 3Novartis Institute for Tropical Diseases, Singapore, Singapore; Introduction
Intracellular pathogens utilize a plethora of approaches to mimic host cell signaling during invasion, parasitic
persistence and replication. Mycobacteria in metabolically inactive and non-replicative states for example pose an
enormous problem in the fight against tuberculosis. A common emerging theme in a wide variety of host-pathogen
interactions is the important regulatory role of lipids in pathogen entry and intracellular trafficking. Phosphoinositides
(PIs, phosphorylated metabolites of phosphatidylinositol, PI) are an important class of membrane lipids and play a role
in a wide variety of cellular processes including signaling, membrane trafficking and cytoskeletal dynamics. They have
been implicated at various steps during bacterial invasion and they also act as ligands of CD1 receptors which present
lipids to the immune system.
We use electrospray ionization mass spectrometry (ESI-MS) to qualitatively and quantitatively profile inositol lipids as
well as other lipids present in complex mixtures derived from mycobacteria in different physiological states (hypoxic
dormancy) or from mammalian cell cultures which were infected with salmonella. The goal of this study is to discovery
pathways that are induced or repressed during adaptation to different growth conditions or during infection.
Chemometric and statistical analysis of mass spectra are used to align and analyze data from replicate experiments.
Differences in lipid profiles are next characterized using tandem mass spectrometry and collision induced dissociation
in order to identify underlying molecular lipid species.
SopB/SigD, a phosphoinositide phosphatase that is delivered into host cells by a type III secretion system, is essential
for the establishment of Salmonella’s intracellular replicative niche. SopB mediates the formation of spacious
phagosomes following bacterial entry and is responsible for the maintenance of high levels of PI(3)P in the bacterial
containing vacuoles. Recombinant SopB preferentially dephosphorylates PI(3,5)P2 and PI(3,4,5)P3 over other
phosphoinositide lipids in vitro. It is attractive to speculate that SopB, through its PI(3,5)P2 phosphatase activity,
mimics the function of Fig4, a Sacharomyces cerevisiae phosphoinositide phosphatase that controls the size and
maturation of the yeast degradative vacuole. Mycobacterial genomes encode for an unusually high number of lipid
enzymes and as a consequence their lipid inventory is accordingly rich and diverse. Lipid extracts from logarithmically
growing and metabolically inactive (dormant) cells were analyzed and compared by thin layer chromatography (TLC)
and electrospray ionization mass spectrometry (ESI-MS). TLC profiles revealed striking differences in regions which
co-migrated with standards for phosphatidylethanolamine (PE) and non-polar esters, such as triacylglycerols (TAG).
ESI-MS indicated a ‘switch’ in abundance of molecular species of PE with different fatty acyl compositions. By tandem
mass spectrometry these ions were identified as PE with palmitic acid (16:0)/oleic acid (18:1), and palmitic
acid/tuberculostearic acid (10-methyl stearic acid), in their chains, respectively. A similar increase in tuberculostearic
acid containing molecular species was observed for phosphatidylinositol (PI). Collectively, these results indicate that
mycobacterium BCG specifically alters part of its lipid inventory upon entry into dormancy. Lipid profiling as described
here is a powerful tool for identification of metabolic pathways, and hence potential enzymes, which are activated in
bacteria that change their physiological states. In addition to promoting our understanding of molecular mechanisms
of infection they also enhance various stages of drug and biomarker development.
LIPID MAPS Eicosanoid Lipidomics: LC-MS Methodologies Enabling the Comprehensive Identification and
Accurate Quantitation of Eicosanoids in RAW 264.7 Cells
Richard Harkewicz; Rebecca C. Bowers-Gentry; Jennifer Cooper; Raymond Deems; Edward A. Dennis; http://www.asms.org/abstracts/DisplayAbstractList.aspx?Session=MODam (3 of 4) [28-May-05 5:32:36 PM] University of California, San Diego, La Jolla, CA Introduction
The LIPID MAPS (Lipid Metabolites And Pathways Strategy) Consortium is an ambitious five-year effort funded by the
National Institute of General Medical Sciences (NIGMS) supporting numerous researchers at 18 universities, medical
research institutes, and companies across the United States working together in a detailed analysis of the structure
and function of lipids.
The LIPID MAPS Eicosanoid Lipidomics Core located at The University of California, San Diego is developing liquid
chromatography-mass spectrometry (LC-MS) based methodologies enabling the comprehensive identification and
accurate quantitation of different lipid eicosanoid classes in a single analysis.
We have compiled an in-house eicosanoid LC-MSMS library from a large number of standards and LIPID MAPS is
making this web-based information freely available to the general lipids community. Our methods have allowed us to
separate and identify thromboxanes, prostaglandins, leukotrienes and the HETE isomers in a single LC-MS analysis
taking less than 16 minutes. Employing isotopic dilution methods, we have accurately quantitated a number of these
eicosanoids in RAW 264.7 cells and have observed a 100 to 400-fold increase in LPS stimulated cells compared to
their basal levels in resting cells. We have also quantitated the level of eicosanoid release as a function of LPS
stimulation time. Chiral chromatography has enabled the determination of eicosanoids produced via enzymatic and
non-enzymatic pathways. In an effort to explore the production of novel eicosanoid species by these cells, we have
subjected them to incubation in deuterium-labeled arachidonic acid and have developed software that efficiently
detects mass-offset analog pairs that indicates an arachidonic acid origin for the compound. During these studies we
have also observed that acid supplementation in the cell growth media results in a two to three-fold increase in
eicosanoid levels. This work is supported by the LIPID MAPS Large Scale Collaborative Grant number GM069338
from the National Institute of Health.
http://www.asms.org/abstracts/DisplayAbstractList.aspx?Session=MODam (4 of 4) [28-May-05 5:32:36 PM]
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