Separation of phospholipids and glycolipids using analytical toroidal-coil countercurrent chromatography. I. Separation of human brain lipids

We demonstrated the separation of human brain lipids using the toroidal-coil countercurrent chromatography (TC-CCC). It became possible to select the suitable two-phase solvent systems, because retention of a stationary phase is much more stable in the TC-CCC than in high-speed countercurrent chromatography (HS-CCC). Optimizing the solvent systems, we succeeded in separating major brain lipids. The two-phase solvent of chloroform: methanol: water (5: 4: 3) was suitable for the separation of acidic phospholipids (phosphatidic acid, phosphatidylserine, phosphatidylinositol, lysophosphatidylinositol, and lysophosphatidylserine). Using hexane: ethylacetate: ethanol: 0.1% aqueous ammonia (5: 5: 5:4), neutral phospholipids (phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine) were separated. Non-polar lipids (cholesterol, alkali-labile glycoglycerolipids and cerebrosides) were separated using the solvent of hexane: ethanol: water (10 : 15: 4). Sphingomyelin (SPM), cerebrosides, and phosphatidylcholine are each reported to have more than 100 molecular species, which are derived from variations of the hydrophobic tail group in mammalian. For this reason, SPM was further separated into two groups (SPM-I and SPM-II). Cerebrosides were separated into several groups using hexane: ethanol: water (5: 4: 3). It was clearly shown that synthesized PC (distearoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol) was completely separated. Phosphatidylserine and phosphatidic acid were also separated in some groups. Because the partition behavior of molecules in the two-phase solvent system can be measured, the TC-CCC could be useful not only for the separation but also for the biological analysis of mammalian cell-membrane lipids.