ABSTRACT
We demonstrate the separation of human brain lipids by using toroidal-coil countercurrent chromatography (TC-CCC). It becomes possible to select the suitable two-phase solvent systems because retention of a stationary phase is much more stable in 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, consisting of chloroform/ methanol/water (5:4:3), was suitable for the separation of acidic phospholipids (phosphatidic acid [PA], phosphatidylserine [PS], phosphatidylinositol [PI], lysophosphatidylinositol [lysoPI], and lysophosphatidylserine [lysoPS]). By using hexane/ethylacetate/etfianol/0.1% aqueous ammonia (5:5:5:4), neutral phospholipids (phosphatidylcholine [PC], sphingomyelin [SPM], and lysophosphatidylcholine [lysoPC]) were separated. Nonpolar lipids (cholesterol, alkali-labile glycoglycerolipids, and cerebrosides [CS]) were separated by 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 tissues. For this reason, SPM was further separated into two groups (SPM-I and SPM-II). Cerebrosides were separated into several groups by using hexane/ethanol/water (5:4:3). It was clearly shown that synthesized PC (distearoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol) was completely separated. Because the partition behavior of molecules in the two-phase solvent system can be measured, TC-CCC could be useful not only for the separation, but also for the biological analysis of mammalian cell-membrane lipids.
