Analysis of ceramides

Quantitation of ceramides and diacylglycerol (DAG) is accomplished most commonly by phosphorylation by the diacylglycerol kinase (DGK) method (9, 10). For the analysis of ceramides one should not use base hydrolysis because this procedure would hydrolyse the DAG. The basis of the assay is the 32P-labelling of the ceramides using a tracer of hot 7-ATP which the kinase uses to phosphorylate the lipids. Once phosphorylated the lipids are re-extracted and then run out on thin layer chromatography (TLC) plates {Figure 5). Detection is done either by film or phosphoimager. Quantitation can be handled either by using Image Quant or by scraping the spots from the plate and doing scintillation counting. The protocol below uses Image Quant analysis to avoid the hazards of scraping (silica and dispersion of radiation).

Figure 5. TIC of ceramide (CER) and diacylglycerol (DAG) post-DGK phosphorylation. Lanes are as follows: lane 1, 160 pmol standards of type III ceramide (Sigma) and diacylglycerot; lanes 2-13, MOLT4 samples; lane 14, 320 pmol standards of type 111 ceramide and diacylglycerol; artd lane 15, 640 pmol standards of type III ceramide and diacylglycerol.

Protocol 6. DGK of ceramides and DAG Reagents

• [i-octyl glucoside, [iOG/dioleoylphos-phatidylglycerol, DOPG (7.5%:2S mM mixed micelle]

Method

1. Construct a standard curve of both ceramide and DAG, including a blank. The range of standards should be 40, 80, 160, 320, 640, and

« chloroform/methanol (1:2) • reaction mix: 103 mM imidazole, 17,9 MgCI;, 2.86 mM dittliothreitol, 1.43 mM EGTA, 71.4 mM LiCI, 71.4 jig/ml DAG kinase, and 0.57 mM diethylenetriamine pentaacetic acid.

1280 pmol. Combining the standards of ceramide and DAG into one tube per pmol amount helps to limit the assay size and the number of lanes to be spotted.

2. Dry down the standards and add to dried samples from either of the aforementioned extraction procedures (Protocols 1 and 2)

3. Resuspend the lipids with 0OG/DOPG and vortex vigorously. If lipids do not go into solution, then sonicate and revortex.

4. Add 70 |xl of reaction mixture to each sample and vortex well.

5. To start the reaction add 10 |xl of ATP with an activity of 4.5 p,Ci per tube.

6. Allow reaction to proceed for 30 min at room temperature.

7. Stop the reaction with the addition of 3 ml of chloroform/methanol and vortex.

8. Add 1 ml of chloroform, 1.7 ml of water, and vortex to induce a phase break (Bligh and Dyer extraction conditions; Protocol 1).

9. Let samples sit briefly, then spin at 2000 gfor 5 min to clarify phases.

10. Carefully aspirate off the upper phase (contains around 90% of the radiation) and collect 1.2-1.5 ml of the lower phase. Dry down the collected lipids via dry nitrogen or a speed vacuum.

11. Resuspend in 50 |xl of chloroform and spot half on TLC plates (Whatman Silica 60A plates).

12. Develop plates in a TLC chamber containing the following solvent system: chloroform/acetone/methanol/acetic acid:water (10:4:3:2:1). Allow tank to equilibrate before running plates.

13. Expose plate to film overnight to obtain an image or directly go to an imager cassette with the phosphor screen and scan in using a phos-phoimager. The resulting scan from the imager may be quantified using Image Quant.

The final quantitation of the ceramides and DAGs can be done after either counts per minute or band intensity have been determined. The actual pico-moles should be obtained from the specific activity of ATP and, therefore, complete conversion of lipids by the kinase is required. Alternatively, quantitation of the mass may be obtained from the standard curve. Since the standard curve is internal and has been through all the experimental manipulations as have the samples, one can directly label them as their starting amounts. The standard curve is also a control for the assay in that it will show whether the assay is in the range of complete phosphorylation, and how well the assay was executed, by its linearity. Once conversion to picomoles has been done this number is for the whole third one started with and can be directly normalized to the third used for lipid phosphate. The minimal change in ceramide usually considered significant is a 50% increase or more and often a rise of twofold or more over basal.

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