Efficient study of sphingolipids in cellular responses entails several basic considerations. The selection of appropriate time points is one of these considerations. Time points should be selected to be relevant to when the biology to be studied is observed. Often this means time points in the range of hours in many cases of sphingolipids in apoptosis; however, shorter time points can be informative for quick changes in sphingolipids. Another basic consideration is the use of time-matched controls from which to compare values from treated samples.
The analysis of various sphingolipid levels requires an initial step for extraction of lipids followed by mild base hydrolysis, which helps by removing most of the glycerolipids whose ester bonds are sensitive to this form of hydrolysis. Quantitation of specific sphingolipids can then be performed using specific and specialized assays. Finally, the levels of sphingolipids are adjusted to either total lipid levels or to cell number.
The starting point for analysis of sphingolipids in apoptosis is the measurement of ceramide levels. When analysing ceramide levels using the diacyl-glycerol kinase assay, care should be exercised in using the enzyme in a quantitative range and not a catalytic range. The reason for starting with ceramide levels is twofold. First, ceramide has been correlated with many cellular events such as differentiation, stress responses, and apoptosis (2). Secondly, changes of ceramide levels are often found in response to stress. An increase in ceramides can result from many pathways, including hydrolysis of sphingomyelin or glycoceramides, inhibition of sphingomyelin synthase or ceramidase, and/or enhancement of de novo synthesis of ceramides. Therefore, once an increase in ceramide is found, further analysis of sphingolipids becomes relevant. The measurement of sphingomyelin can show whether the increase in ceramide levels is from its hydrolysis or another source. Sphingomyelin hydrolysis has been implicated in some ceramide increases, as represented by the SM cycle (2). Sphingoid backbone levels have not been implicated to the same extent as sphingomyelin and ceramide levels. Enhancement of sphingolipid turnover studies can be gained by analysis of the SMases and SM synthase. Changes in the activity of these enzymes reflect either an induction in enzyme amounts and/or some poorly defined form of post-translational modification. Neither the acid/neutral SMase assay or SM synthase assay will detect reversible allosteric regulation of the enzymes. Together, the above analyses allow for an initial in depth look at the possible role of sphingolipids in a given cellular response.
3, Extraction of sphingolipids and normalization by lipid phosphate
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