ANALYSIS OF CERAMIDES
Ceramides may be found in free form or included in more complex lipids such as sphingomyelin or various glycosphingolipids. Analysis of the ceramide moieties of these complex lipids makes it possible to determine their individual molecular species since Ceramides are combinations of both a fatty acid and a long-chain base.
Free ceramides which are most frequently present in trace amounts in biological extracts must be derivatized directly before separation and HPLC analysis. When ceramides are prepared from phospholipids or glycosphingolipids (in the range 0.1-10 mg), they are more conveniently purified by column chromatography before further analysis.
The lipidologist may want to quantify or to know the structure of ceramides. In all cases, their purification from the whole bulk of lipids is necessary, either before or after derivatization, to allow their quantification. This quantification may be carried out by the estimation of ceramide components (fatty acids, bases) or directly by the detection of UV absorbing or fluorescent tags.
According to the aim of the study, three questions may be defined:
How to separate and prepare ceramides ?
How to quantify a pool of ceramides ?
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