2. Michel, C., van Echten-Deckert, G., Rother, J., et al. Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide. J. Biol. Chem. 272(36), 22432-22437 (1997). 

3. Ji, L., Zhang, G., Uematsu, S., et al. Induction of apoptotic DNA fragmentation and cell death by natural ceramide. FEBS Lett. 358(2), 211-214 (1995). 

4. Norm Radin (unpublished observations)

Sphingolipid Isolation

Due to the wide range of physical properties between various lipid species, it is difficult to isolate all lipids with one technique. Different processes are generally used to extract polar and non-polar compounds. Some methods allow for the isolation of all lipid species but are not necessarily as quantitative as more specialized protocols. Many references are available to choose from depending on your requirements. Two very helpful online resources to get familiar with various protocols are available at the AOCS Lipid Library and Cyberlipid Center.

The most common extraction techniques for lipids are the Folch and the Bligh and Dyer methods, which are outlined below:

Folch Method

1. Homogenize tissue sample with chloroform/methanol 2:1, 20 ml/gram of tissue.

2. Remove the solids by filtration or centrifugation.

3. Add 0.2 volumes of 0.9% KCl or NaCl.

4. Gently mix and allow the layers to separate. Centrifugation will speed up the separation.

5. Gangliosides are mostly present in the aqueous phase and can be recovered by reversed phase chromatography.

6. Almost all other lipids are in the organic solvent layer and can be concentrated and purified by normal phase chromatography. 
   
   

Bligh and Dyer Method

1. Homogenize tissue sample with 1 ml of water, 1M NaCl, or 1 M KCl.

2. Add 3.75 ml chloroform/methanol 1:2 and homogenize.

3. Add 1.25 ml chloroform and mix.

4. Add 1.25 ml water and mix.

5. Centrifuge to separate the aqueous and organic phases.

6. Gangliosides are mostly present in the aqueous phase and can be recovered by reversed phase chromatography.

7. Almost all other lipids are in the organic solvent layer and can be concentrated and purified by normal phase chromatography.
   
   

Suggested References for Analytical Sphingolipid Extraction and Analysis

Glucosylsphingosine

Fuller, M., Szer, J., Stark, S., et al. Rapid, single-phase extraction of glucosylsphingosine from plasma: A universal screening and monitoring tool. Clin. Chim. Acta 450, 6-10 (2015). 

Sulfatides

Barcenas, M., Suhr, T.R., Scott, C.R., et al. Quantification of sulfatides in dried blood and urine spots from metachromatic leukodystrophy patients by liquid chromatography/electrospray tandem mass spectrometry. Clin. Chim. Acta 433, 39-43 (2014).

Gangliosides

Masson, E.A.Y., Sibille, E., Martine, L., et al. Apprehending ganglioside diversity: A comprehensive methodological approach. J. Lipid Res. 56(9), 1821-1835 (2015).

Zhang, Y., Wang, J., Liu, J. et al. Combination of ESI and MALDI mass spectrometry for qualitative, semi-quantitative and in situ analysis of gangliosides in brain. Sci. Rep. 6, 25289 (2016).

Tropak, M.B., Yonekawa, S., Karumuthil-Melethil, S., et al. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo. Mol. Ther. Methods Clin. Dev. 3, 15057 (2016).

Fatty Acid Analysis of Glycerides, Phospholipids, and Sphingolipids by GC/FID

Natural sphingolipids contain a heterogeneous mixture of fatty acids attached to the sphingosine backbone. The fatty acid composition is dependent on several factors including species, location within the organism, age, and environmental conditions. Normal variations in the fatty acids include chain-length, hydroxylation, and unsaturation. To determine the fatty acid components present in a sphingolipid sample, the fatty acid is hydrolyzed from the sphingosine backbone and then converted to a fatty acid methyl ester. It can then be analyzed by GC using flame ionization detection (FID), and the individual fatty acid methyl esters identified by comparison of the retention times to reference standards.

Preparation of Methyl Esters from Glycerides and Phospholipids

Methanolic Base Hydrolysis

1. Weigh 5 mg of dry sample to be tested into a reaction vessel.

2. Add 1 ml of hexane to the sample.

3. Add 50 μl of 1 M sodium methoxide in methanol.

4. Mix thoroughly.

5. Let sit at room temperature for 5 minutes.
   

6. The sample is now ready to analyze by GC/FID.
   
   

Preparation of Methyl Esters from Phospholipids, Neutral Sphingolipids, Neutral Glycosphingolipids, and Acidic Glycosphingolipids

Methanolic Sulfuric Acid

1. Make a solution of 2% sulfuric acid in methanol. The 2% sulfuric acid/methanol solution must be made fresh before use.

2. Add 1 ml of the sulfuric acid solution to the dry lipid sample in a reaction tube.

3. Blanket with argon and seal with a Teflon-lined cap.

4. Place in an equilibrated heating block as indicated below:

1. Fatty acid to methyl ester – 5 minutes at 80°C

2. 2- or 3-Hydroxy fatty acids – 1.5 hours at 80°C  

3. Phospholipids – 2 hours at 70°C

4. Glycolipids – 6 hours at 100°C

5. Sphingomyelin – 2 hours at 100°C

5. At the end of the desired time, remove from the heating block and let cool to room temperature. Do not open until cool!

6. Add 0.5 ml of deionized water, which will stop the reaction.

7. Add 4 ml of hexane and shake vigorously.
   

8. Let stand until two phases are visible. Note: Top hexane layer must be clear.

9. Remove the top hexane layer and save.

10. Extract the bottom layer by repeating steps 7-9 two more times.

11. Discard the bottom layer after fully extracted.

12. Combine the three top layers and add a 0.5-1 g of a mixture of sodium sulfate/ sodium bicarbonate (4:1).

13. Shake vigorously.

14. Using a pipette, transfer the sample to a reaction tube. Note: Be careful not to pull any of the sodium sulfate/sodium bicarbonate solids.

15. Concentrate with a stream of nitrogen down to 1 ml.

16. The sample is now ready to analyze by GC/FID.
    
    

GC/FID Analysis of Fatty Acid Methyl Esters

GC Conditions*

1. Column: SP-2330 or RTX-2330; 30 x 0.25 mm x 0.2 μm

2. Oven: 200°C Isothermal

3. Carrier Linear Velocity: Helium at 20 cm/sec.

4. Detector: FID, 250°C
   

5. Injector: 250°C
   

6. Split ratio: 100:1

   
   *These are general GC conditions for fatty acid methyl esters and may not separate all components of a sample.

Storage and Handling of Lipids

Natural lipid products can contain significant amounts of unsaturation, both in the fatty acid moieties and elsewhere. These are prone to autoxidation when exposed to oxygen, water, or other oxidants. Temperature can also significantly impact the rate of oxidation. Unsaturated lipids should be handled with extra precautions.

Catalog items in unopened containers are generally stable for at least one year when stored under the conditions indicated in the catalog listing. Items containing unsaturated fatty acids are especially subject to oxidation and should be stored in a solution of organic solvents, or under argon, at -20°C. Minimize exposure to air for these products. Glycolipids and phospholipids should not be stored in aqueous solutions due to potential hydrolysis.

Never use plastic or polymer materials with organic solvents as doing this will leach out impurities from the material. Always use glass, stainless steel, or Teflon equipment for transferring lipids in organic solvents and for storage. Lipids should always be stored in glass containers with Teflon lined caps.

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