Quantitative Determination of Ceramide Molecular Species in Dendritic Cells

The activation of acid sphingomyelinase by cellular stress or receptors or the de novo synthesis lead to the formation of ceramide (N-acylsphingosine), which in turn modifies the biophysical properties of cellular membrane and greatly amplifies the intensity of the initial signal. Ceramide, which acts by re-organizing a given signalosome rather than being a second messenger, has many functions in infection biology, cancer, cardiovascular syndromes, and immune regulation. Experimental studies on the infection of human cells with different bacterial agents demonstrated the activation of the acid sphingomyelinase/ceramide system. Moreover, the release of ceramide was found to be a requisite for the uptake of the pathogen. Considering the particular importance of the cellular role of ceramide, it was necessary to develop sensitive and accurate methods for its quantification.

Here, we describe a method quantifying ceramide in dendritic cells and defining the different fatty acids (FA) bound to sphingosine. The main steps of the method include extraction of total lipids, separation of the ceramide by thin-layer chromatography, derivatization of ceramide-fatty acids (Cer-FA), and quantitation of these acids in their methyl form by gas chromatography on polar capillary columns. The identification of FA was achieved by means of known standards and confirmed by mass spectrometry.

FA ranging between C10 and C24 could be detected and quantified. The concentration of the sum of Cer-FA amounted to 14.88 ± 8.98 nmol/106 cells (n=10). Oleic acid, which accounted for approximately half of Cer-FA (7.73 ± 6.52 nmol/106 cells) was the predominant fatty acid followed by palmitic acid (3.47 ± 1.54 nmol/106 cells).

This highly sensitive method allows the quantification of different molecular species of ceramides.