When tissues are rich in highly polar lipids such as gangliosides, a reliable extraction method is needed to prevent their loss while extracting all other lipid classes. All the previously described techniques brings gangliosides and likely a part of other very polar lipids into the water-rich layer. A method, recently described for nervous tissues (Dreyfus et al., Anal Biochem 1977, 249, 67-78), prevents these drawbacks.
Small tissue samples corresponding up to 10 mg protein suspended in 0.5 ml water are mixed with 5 ml chloroform/methanol mixture (1/1) for 30 min. The pellet obtained by centrifugation is extracted again with successively 3 ml of the same solvent, 3 ml of a mixture chloroform/methanol (1/2) and 3 ml of a mixture chloroform/methanol/water (60/30/4.5). The four lipid extracts are combined and evaporated and the dry residue dissolved for further purification.
A liquid/liquid extraction has been used with success for mass spectroscopy estimation of brain gangliosides (Garcia AD et al., J Chromatogr B 2014, 947-8, 1). The lipid extract was suspended in a mixture of chloroform/methanol/water (30/60/8, v/v/v), vortexed and sonicated. The solution was then centrifuged and the aqueous phase containing the enriched gangliosides was collected and set aside while the organic phase was then subjected to the same extraction procedure. The supernatants were combined and dried under vacuum. Once dry, the lipid extracts were resuspended in 10 mL of the liquid chromatography starting buffer. The gangliosides were purified on C8 SPE cartridges before LC-MS analysis.
A very efficient method has been developed to isolate and purify GM1 from pig brain (Bian L et al., Biomed Chromatogr 2015, 29, 1604). The method consisted of a precipitation by acetone followed by an extraction by chloroform–methanol–water. The purification was done using a two-step chromatographic separation by DEAE–Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The final yield of GM1 was about 0.022% ( g/g) with the purity of about 98%. This method will probably provide a reference alternative for isolation and purification of other amphipathic substances in biological tissues.
Lyso derivatives (N-deacylated) of glycosphingolipids are not efficiently recovered from cell extracts because of their high polarity. The glycosylsphingosine are found in tissues from patients and in animal models. In normal conditions, their concentration are very low and needs very efficient techniques to be evaluated. It was shown that a second extraction with water-saturated butanol of the upper aqueous phase obtained after a Folch extraction was necessary to recover up to 98% of the tissue lysoglycosphingolipids (Bodennec J et al., J Lipid Res 2003, 44, 218).