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Journal of Lipid Research, Vol. 43, 510-522, March 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography

Claude Vieua, François Tercéa, Françoise Chevyb, Corinne Rollanda, Ronald Barbarasa, Hugues Chapa, Claude Wolfb, Bertrand Perreta, and Xavier Colleta
a Institut Fédératif de Recherche Claude de Preval, IFR 30, Institut National de la Santé et de la Recherche Médicale, Unité 326, Phospholipides membranaires, Signalisation cellulaire et Lipoprotéines, Hôpital Purpan, 31059, Toulouse Cedex, France
b Laboratoire Commun de Spectrométrie de masse, Faculté de médecine de St Antoine, Université de Paris 6, Paris, France

Correspondence to: Xavier Collet, To whom correspondence should be addressed., collet{at}cict.fr (E-mail)

This study reports a single-step analysis of the molecular species of endogenous ceramides and of the ceramide moiety of sphingomyelins in biological samples, using gas liquid chromatography (GLC). Silylated sphingomyelins were quantitatively converted to monosilylated ceramide upon injection into GLC, whereas the free ceramides were di-silylated on the primary and secondary alcohol function, as confirmed by mass spectrometry. The reproducible shift of the retention times between the mono- and di-silylated derivatives enables simultaneous quantification of the variety of sphingomyelin and ceramide molecular species. Overlapping diacylglycerols were first removed by a mild alkaline treatment of the lipid extract. The lowest detection limit (5 pmol) did not allow for identification of free ceramides in human plasma, but 17 molecular species of ceramides derived from sphingomyelins were quantified, from NC16:0 up to NC24:1. By contrast, three major free ceramides (NC16:0, NC24:0, and NC24:1) were quantified in HEPG2 and Chinese hamster ovary (CHO) cells. Upon induction of apoptosis in CHO cells by C6-ceramide, we could follow the disappearance of the C6-ceramide, its partial conversion to C6-sphingomyelin, and the prominent increase of NC16:0 ceramide.

Thus, our method represents a unique procedure of simultaneous analysis of sphingomyelin and ceramide molecular species able to monitor the variation of the different pools in biological samples. — Vieu, C., F. Tercé, F. Chevy, C. Rolland, R. Barbaras, H. Chap, C. Wolf, B. Perret, and X. Collet. Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography. J. Lipid Res. 2002. 43: 510–522.

Supplementary key words: mass spectrometry, apoptosis, lipid analysis, plasma, CHO cells, HepG2 cells


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