PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation[S] 
Journal of Lipid Research  2013;54(11):3085-3097.
Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.
doi:10.1194/jlr.M041533
PMCID: PMC3793613  PMID: 23883581
Oxidized phospholipids; atherosclerosis; PGE2/D2-PEs
2.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
doi:10.1074/mcp.M900275-MCP200
PMCID: PMC2849712  PMID: 20037178
3.  Targeted Lipidomic Analysis of Oxysterols in the Embryonic Central Nervous System 
Molecular bioSystems  2009;5(5):529-541.
Summary
In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC) – mass spectrometry (MS) and tandem mass spectrometry (MSn) low levels of oxysterols (estimated 2 – 165 ng/g wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7α-, 7β-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 μg/g in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng/g in Ctx and 13 ng/g in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng/g in Ctx and 91 ng/g in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg/g in Ctx and 1.15 mg/g in Sc) and its precursors were determined by gas chromatography – mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
doi:10.1039/b819502a
PMCID: PMC2702751  PMID: 19381367
4.  Discovering Oxysterols in Plasma: A Window on the Metabolome 
Journal of proteome research  2008;7(8):3602-3612.
While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible “window” to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome i.e. oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography – mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7α-, 24S- and 27-hydroxycholesterol; the cholestenetriol 7α,27-dihydroxycholesterol; and 3β-hydroxycholest-5-en-27-oic acid and its metabolite and 3β,7α-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma i.e. cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6-seco-sterols and steroid hormones. Although involving derivatisation, sample preparation is straight forward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols and steroid hormones opening a “window” to view this region of the metabolome.
doi:10.1021/pr8001639
PMCID: PMC2567817  PMID: 18605750
metabolomics; lipidomics; steoidomics; sterols; steroids; high-performance liquid chromatography – mass spectrometry; tandem mass spectrometry; electrospray; ion-trap; derivatisation
5.  Potential of sterol analysis by liquid chromatography - tandem mass spectrometry for the prenatal diagnosis of Smith-Lemli-Opitz syndrome 
Clinical chemistry  2008;54(8):1317-1324.
Background
Smith-Lemli-Opitz syndrome (SLOS) is a severe disorder of cholesterol synthesis classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. In recognition of the current move towards tandem mass spectrometry (MS/MS) methodologies we present prototype LC-MS/MS methods for the accurate diagnosis of the disorder
Methods
3β-Hydroxysterols in amniotic fluid are oxidised with cholesterol oxidase to their corresponding 3-ketones, which are then derivatised with Girard P (GP) hydrazine in a “one-pot” reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS due to the presence of a charged quaternary nitrogen and are analysed by reversed-phase LC-ES-MS/MS. Both capillary LC-MS/MS and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper absorbed blood spots.
Results
In a double blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the 7+8-dehydrocholesterol (7+8-DHC) to cholesterol ratio was found to lie below 0.02 (range, 0.00 - 0.02: mean ± SD, 0.01 ± 0.007) in all control samples (intra assay variation 5.91%), and above 0.20 (range, 0.20 - 1.13: mean ± SD, 0.79 ± 0.35) in SLOS (intra assay variation 4.56%), corresponding to a difference in ratios between the two groups of a factor of at least 10. The limit of quantification was equivalent to 2 nL of amniotic fluid injected on-column.
Conclusion
Here we describe a “proof-of-concept” study for the prenatal diagnosis of SLOS. However, further developments will be necessary to automate sample handling and reduce chromatographic time in order to allow the methodology to be used for pre- and postnatal diagnosis.
doi:10.1373/clinchem.2007.100644
PMCID: PMC2533047  PMID: 18556335
6.  Matrix-Assisted Laser Desorption/Ionisation - High-Energy Collision-Induced Dissociation of Steroids: Analysis of Oxysterols in Rat Brain 
Analytical chemistry  2006;78(1):164-173.
Neutral steroids have traditionally been analysed by gas chromatography – mass spectrometry (GC-MS) after necessary derivatisation reactions. However, GC-MS is unsuitable for the analysis of many conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3β-hydroxy-Δ5 or 5α-hydrogen-3β-hydroxy structure. Steroids were derivatised with Girard P (GP) hydrazine to give GP hydrazones which are charged species and readily analysed by matrix-assisted laser desorption/ionization mass spectrometry. The resulting [M]+ ions were then subjected to high-energy collision-induced dissociation on a tandem time-of-flight instrument. The product-ion spectra give structurally informative fragment-ion patterns. The sensitivity of the analytical method is such that steroids structures can be determined from low pg (low fmole) amounts of sample. The utility of the method has been demonstrated by the analysis of oxysterols extracted from rat brain.
doi:10.1021/ac051461b
PMCID: PMC2315783  PMID: 16383324
7.  Liquid Chromatography Combined with Mass Spectrometry Utilising High-Resolution, Exact Mass, and Multi-Stage Fragmentation for the Identification of Oxysterols in Rat Brain 
Journal of lipid research  2007;48(4):976-987.
In man the brain accounts for about 20% of the body's free cholesterol, most of which is synthesised de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolised to 24S-hydroxycholesterol principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover, however, the route by which the remainder is turned over has yet to be elucidated. Here we describe a novel liquid chromatography (LC) – multi-stage fragmentation mass spectrometry (MSn) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatisation to enhance ionisation, exact mass analysis at high-resolution to identify potential metabolites, and LC-MS3 to allow their characterisation. 24S-Hydroxycholesterol was confirmed as a major oxysterol in rat brain, while other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24, 7α,25-, and 7α,27-dihydroxycholesterols. In addition, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterised in rat brain.
doi:10.1194/jlr.M600497-JLR200
PMCID: PMC2315781  PMID: 17251593
Sterol; Cholesterol; 24S-hydroxycholesterol; dihydroxycholesterol; secosterol; derivatisation; Girard P; LTQ Orbitrap; Liver X Receptor; Alzheimer's disease

Results 1-7 (7)