Isolation of Sphingolipids for Analysis

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 the researchers requirements. Two very helpful online resources to get familiar with various protocols are available at the AOCS Lipid Library and Cyberlipid.

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.
    • Gangliosides can be recovered by reversed phase chromatography.
  6. Almost all other lipids are in the organic solvent layer.
  7. Concentrate and purify by normal phase chromatography.

Bligh and Dyer Method:
  1. Homogenize tissue sample with 1 ml of water or 1M NaCl or 1M KCl.
  2. Add 3.75 ml choroform/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.
    • Gangliosides can be recovered by reversed phase chromatography.
  7. Almost all other lipids are in the organic solvent layer.
  8. Concentrate and purify by normal phase chromatography.

References for Analytical Sphingolipid Extraction and Analysis:
  1. Glucosylsphingosine: M. Fuller et al., Rapid, single-phase extraction of glucosylsphingosine from plasma: A universal screening and monitoring tool. Clin. Chim. Acta. 460, 6-10 (2015) doi: 10.1016/j.cca.2015.07.026
  2. Sulfatides: M. Barcenas 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) doi: 10.1016/j.cca.2013.12.016
  3. Gangliosides:
    1. E. Masson et al., Apprehending ganglioside diversity: a comprehensive methodological approach. J. Lipid Res. 56(9), 1821-1835 (2015) doi: 10.1194/jlr.D060764
    2. Z. Yangyang 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) doi: 10.1038/srep25289
    3. M. Tropak 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) doi: 10.1038/mtm.2015.57