High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids

Kessel, W.S.M. Geurts van; Hax, W.M.A.; Demel, R.A.; Gier, J. De

doi:10.1016/0005-2760(77)90102-3

Cited by 228 publications

(40 citation statements)

References 21 publications

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“…Fractionation of bacterial lipids by high-performance liquid chromatography (HPLC) was accomplished using a semipreparative HPLC column (1 ϫ 25 cm silica gel, 5 m; Supelco, Inc., Bellefonte, PA) and eluted isocratically with hexane-isopropanolwater (6:8:0.75; v/v/v: solvent A) (8). Lipid samples were dissolved in solvent A to achieve a concentration of ‫ف‬ 80 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis

Nichols

Riep

Mun

et al. 2004

Journal of Lipid Research

143

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Porphyromonas gingivalis , a recognized periodontal pathogen, synthesizes free ceramides as well as other phosphorylated ceramide lipids. The purpose of this study was to separate complex lipids of P. gingivalis by high-performance liquid chromatography (HPLC) and determine the structures and biological activities of the major ceramide classes. Using gas chromatography-mass spectrometry, electrospray tandem mass spectrometry (ESI-MS/MS) and NMR analyses, three major classes of dihydroceramides were identified in specific HPLC fractions, with all classes containing the same dihydroceramide base structures (

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Section: Methodsmentioning

confidence: 99%

Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis

Nichols

Riep

Mun

et al. 2004

Journal of Lipid Research

143

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“…Lipid-free controls containing this amount of solvent were tested and did not alter EC migration. Extracted lipids were dried under N 2 , dissolved (at 4 mg/ml) in n -hexane:2-propanol:water (39:52:9, vol/vol), and fractionated (1 ml/min) on a silica HPLC column (Lichrosorb SI-60 10 m; 250 ϫ 4.6 mm; Alltech Associates Inc., Deerfield, IL) using the same solvent under isocratic conditions (38). Eluted lipids were detected by absorbance at 206 nm.…”

Section: Methodsmentioning

confidence: 99%

Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

Murugesan¹,

Fox²

1996

J. Clin. Invest.

104

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Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a halfmaximal activity at 12-15 M, and near complete inhibition at 20 M; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPC's containing 1-position C 16 or C 18 saturated fatty acids were antimigratory, but those containing C Յ 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A 2 (PLA 2 ). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract confirming that lysoPC (or a co-eluting lipid) was a major antimigratory molecule in oxidized LDL. These studies demonstrate that lysoPC

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“…Postnuclear supernate (2 ml) fractions were combined and layered on top of 30% Percoll (Sigma-Aldrich) in Buffer A and centrifuged for 30 min at 84,000 g in a Beckman Ti60 rotor. The plasma membrane fraction, a visibly cloudy band ‫ف‬ 5.7 cm from the bottom using modifi cations of previously described methods ( 37 ). Samples were dried down in a stream of argon, and the residue was dissolved in 100 µl of chloroform-methanol (2:1) and 60 µl injected onto a 3.9 × 300 mm Waters µPorasil column (10 µm particle size).…”

Section: Detergent-free Membrane Isolationmentioning

confidence: 99%

Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers

Sorci‐Thomas

Owen

Fulp

et al. 2012

Journal of Lipid Research

108

147

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In humans, the plasma concentration of HDLs has been repeatedly shown to be inversely correlated with the risk of developing coronary heart disease ( 1, 2 ). The concentrations of plasma HDL has been shown to be largely dependent on hepatic ATP-binding cassette transporter A1 (ABCA1) ( 3-5 ), which transports and promotes the effl ux of glycerophosphocholine (PC), free cholesterol (FC), and sphingomyelin (SM) to wild-type apolipoprotein A-I (apoA-I), resulting in the formation of nascent HDLs (nHDLs). Functional mutations in human ABCA1 cause Tangier disease ( 6, 7 ), which is characterized by very low levels of plasma HDL apoA-I. Tangier disease is believed to alter a process termed "reverse cholesterol transport." Although defects in ABCA1 function have been identifi ed by Abstract This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ‫ف‬ 12, 10, 7.5, and <6 nm) and composition were purifi ed by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ‫ف‬ 12 nm and 10 nm diameter, contained ‫ف‬ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ‫ف‬ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1 . -Sorci-Thomas, M. G., J. S. Owen, B. Fulp, S. Bhat, These studies were supported by grants from the National Institutes of Health grants and Abbreviations: BMDM, bone marrow-derived macrophage; CE, cholesteryl ester; DSP, dithiobis(succinimidylpropionate) ; EM, electron microscopy; FC, free cholesterol; FPLC, fast protein liquid chromatography; GP, glycerophospholipids; HEK, human embryonic kidney; HexCer; hexosylceramides; LPC, lyso-glycerophosphocholine; MSCE, mass spectrometer collision energy; NDGGE, nondenaturing gradient gel electrophoresis; nHDL, nascent HDL; PC, glycerophosphocholine; PE, glycerophospho ethanolamine; PG, glycerophosphoglycerol; PI, glyerophos phoinositol; PM, plasma membrane; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, glycerophosphoserine; rHDL, recombinant HDL; RT, room temperature; SL, sphingolipid; TC, total cholesterol.1 To whom correspondence should be addressed. e-mail...

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High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids

Cited by 228 publications

References 21 publications

Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis

Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis

Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers

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