High-performance liquid chromatographic separation and photometric detection of phospholipids

Hax, W.M.A.; Kessel, W.S.M. Geurts van

doi:10.1016/s0021-9673(01)92081-3

Cited by 127 publications

(24 citation statements)

References 19 publications

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“…Ligands were extracted from the CD1d molecules using the chloroform/methanol/water method described by Bligh and Dyer [25]. Lipid classes were separated by normal phase-HPLC (Luna 3 μ Silica (2) 100 Å, 150×2 mm column, Phenomenex, Torrance, CA) using a gradient of n-hexane:2-propanol:water (41:54:5 v/v) and n-hexane:2-propanol:water (37:54:9 v/v), as previously described [38]. Fractions of 200 µL were collected at 1.0 min intervals and monitored by UV absorption at 210 nm.…”

Section: Methodsmentioning

confidence: 99%

Determination of Cellular Lipids Bound to Human CD1d Molecules

et al. 2009

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CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death.

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Section: Methodsmentioning

confidence: 99%

Determination of Cellular Lipids Bound to Human CD1d Molecules

et al. 2009

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“…a small number of samples were also separated by h.p.l.c. (Hax & Guerts van Kessel, 1977). Polyphosphoinositides were separated by chromatography on immobilized neomycin (Schacht, 1978) or, when separation of lysophosphoinositides was also required, by chromatography on silica gel H (Analtech) using the solvent system chloroform/methanol/4M-NH40H (9:7:2, by vol.)…”

Section: Methodsmentioning

confidence: 99%

The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes

Meade¹,

Turner²,

Bateman³

1986

Biochemical Journal

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Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products oftheir breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a 'second signal' (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.

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“…Phospholipids do not have specific UV absorption peaks, but display strong absorption in the 203-214 nm region, reflecting a combination of unsaturated carbons, carbonyl, carboxyl, phosphate, amino and quaternary ammonium groups (Hax & Geurts van Kessel, 1977).…”

Section: Separation and Detection Of Lipids By Hplcmentioning

confidence: 99%

Organic phosphorus in marine sediments : chemical structure, diagenetic alteration, and mechanisms of preservation

Laarkamp¹

2000

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Phosphorus, an essential nutrient, is removed from the oceans only through burial with marine sediments. Organic phosphorus (Po,g) constitutes an important fraction (ca. 25%) oftotal-P in marine sediments. However, given the inherent lability of primary Po,g biochemicals, it is a puzzle that any Pm, is preserved in marine sediments. The goal of this thesis was to address this apparent paradox by linking bulk and molecular-level Po" information.A newly-developed sequential extraction method, which isolates sedimentary Po" reservoirs based on solubility, was used in concert with 3lp nuclear magnetic resonance spectroscopy (31p_NMR) to quantify Po,g functional group concentrations. The coupled extraction/ 31 P-NMR method was applied to three sediment cores from the Santa Barbara Basin, and the first-ever high-resolution depth profiles of molecular-level Po,g distribution during diagenesis were generated.These depth profiles were used to consider regulation of Pm, distribution by biomass abundance, chemical structure, and physical protection mechanisms. Biomass cannot account for more than a few percent of sedimentary Po,g. No evidence for direct structural control on remineralization of Po,g was found. Instead, sorptive protection appears to be an important mechanism for Po,g preservation, and structure may act as a secondary control due to preferential sorption of specific Pm, compound classes.

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High-performance liquid chromatographic separation and photometric detection of phospholipids

Cited by 127 publications

References 19 publications

Determination of Cellular Lipids Bound to Human CD1d Molecules

Determination of Cellular Lipids Bound to Human CD1d Molecules

The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes

Organic phosphorus in marine sediments : chemical structure, diagenetic alteration, and mechanisms of preservation

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