Separation and Purification of Sphingomyelin Diastereomers by High-Performance Liquid Chromatography

Ramstedt, Bodil; Slotte, J. Peter

doi:10.1006/abio.2000.4612

Cited by 9 publications

(8 citation statements)

References 27 publications

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“…59 pmol/nmol of total lipid). Similar to other studies, we observed that SM separated in two bands by TLC [24,25]. The lower band represents short-chain (C16:0) and the upper band long-chain (C24:0/1) SM species.…”

Section: Apoptosis Sensitivity Of Wtnih3t3 Spiβ and Spiβs262a Cellssupporting

confidence: 91%

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

Schenning

Tiel

Wirtz

et al. 2007

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPbeta is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells.

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Section: Apoptosis Sensitivity Of Wtnih3t3 Spiβ and Spiβs262a Cellssupporting

confidence: 91%

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

Schenning

Tiel

Wirtz

et al. 2007

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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“…LC has the additional advantages that it a ) reduces the complexity of the electrospray droplet, thereby decreasing the likelihood that it will be composed of compounds with greatly different gas phase basicity or acidity, which can suppress the ionization of the analyte(s) of interest ( 42 ); b ) separates salts in the biological extract from the compounds of interest; hence, it minimizes formation of multiple salt adducts and other sources of ionization suppression ( 43 ); c ) concentrates the analytes of interest in a small volume; and d ) with appropriate choice of LC conditions, separates isomers that might not be distinguished by MS or MS/MS alone ( 44,45 ).…”

Section: Discussionmentioning

confidence: 99%

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Shaner

Allegood

Park

et al. 2009

Journal of Lipid Research

384

366

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“…Additionally, LC serves to concentrate the analytes of interest into a small elution volume thereby increasing sensitivity. Finally, LC may separate isomers (with appropriate choice of separation conditions) that might not be distinguished by MS or MS/MS alone [61, 62]. …”

Section: Discussionmentioning

confidence: 99%

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sullards

Liu

Chen

et al. 2011

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

126

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Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.

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Separation and Purification of Sphingomyelin Diastereomers by High-Performance Liquid Chromatography

Cited by 9 publications

References 27 publications

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

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