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

Abstract

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 disilylated 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.

Metabolite Signatures of Metabolic Risk Factors and their Longitudinal Changes

“…Compounds were de-tected either underivatized, oximated and silylated, or silylated. Some lipid compounds, eg, sphingomyelins, were detected as a thermal degradation product by losing the phosphocholine group in the injector (19). Metabolites (known or unknown) specific for this study were added to the target table by searching the first three batches of samples for peaks present but not accounted for by the preselected target table.…”

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

“…To this end, cells expressing either probe were treated for 2 h with 200 μM of the ceramide analogue C6-ceramide [ 64 ] or overnight with 200 μM of the glucosyl-ceramide synthase inhibitor Genz-123346 [ 65 ] or their respective ethanol and methanol vehicle controls. Previously, C6-ceramide has been shown to induce large increases in endogenous ceramides and hexosyl-ceramides, with an only comparatively smaller increase in sphingomyelin [ 64 , 66 , 67 ]. In contrast, Genz-123346 increased the ceramides without affecting sphingomyelin and concomitantly decreasing hexosyl-ceramides [ 65 , 68 ].…”

“…The effects of Fum (fumonisin B1), C6 (C6-ceramide), and Genz (Genz-123346) were extrapolated from the literature. ( 1 [ 50 ]; 2 [ 66 ]; 3 [ 67 ]; 4 [ 68 ]). ( B ) Hypothetical model of the behavior of N-KSR, C-KSR, and C-KSR-GS before and during phagocytosis progression (blue arrows).…”

Development of genetically-encoded fluorescent KSR1-based probes to track ceramides during phagocytosis

“…The effects of Fum (fumonisin B1), C6 (C6-ceramide) and Genz (Genz-123346) are extrapolated from the literature. ( 1 Merrill et al, 2011; 2 Vieu et al, 2002; 3 Duran et al 2012; 4 Koike et al, 2019). B) Hypothetical model of the behavior of N-KSR, C-KSR and C-KSR-GS before and during phagocytosis.…”