Analysis of sphingoid backbones and sphingomyelins requires an additional step of refinement. This step is a mild alkaline hydrolysis, which is useful for hydrolysing most glycerolipids, leaving the resistant sphingolipids. Mild alkaline hydrolysis is not used for ceramide measurement when there is additional interest in diacylglycerol, which is sensitive to the procedure. Thus, one can quickly and easily purify the organic lipid extract to one containing mainly sphingolipids.
Protocol 3. Mild alkaline hydrolysis of lipids
1. Resuspend dried down lipids from either the Bligh and Dyer (Protocol 1) or Folch (Protocol 2) extractions in 2 ml of chloroform.
2. Add 0.2 ml of KOH in methanol to the tubes and vortex vigorously.
3. Incubate in a shaking water bath at 37°C for 1 h.
4. Immediately add 0.2 ml of 2 N HCI in methanol to neutralize the KOH.
5. Now add 0.6 ml of methanol and 0.4 ml of water and vortex to break the solution into an organic (lower) and aqueous (upper) phases. The lower phase contains the alkaline-resistant sphingolipids, while the upper has the other hydrolysed lipids.
6. Aspirate off the upper, aqueous phase. Wash the organic phase with 1 ml of pH-neutral water (make neutral by use of 50 |xl of 1 N NH4OH per 15 ml of distilled water). Vortex after adding the water and let the samples set for 15 min. Spin at 2000 g in a table-top centrifuge to clarify the phase break then aspirate off the aqueous phase. Repeat wash as above.
7. Dry down the organic phase under nitrogen or by a speed vacuum.
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