Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids.
Quantitative separation of ceramide, sphingoids (dihydrosphingosine, sphingosine, psychosine), and glycosphingolipids as individual fractions was achieved with silicic acid, Dowex column chromatography, and specific solvent mixtures that have not been previously described. Purified ceramide, resolved as a single band, was assayed by thin-layer chromatography (TLC) followed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Sphingoids, purified by Dowex, were assayed by GC and HPLC without mild alkaline hydrolysis, which reduces the yield by interfering with the free amino group of psychosine and dihydrosphingosine. Several less polar (than cerebroside) alkali-/acid-labile glycosphingolipids that elute with galactosylceramide were also identified. Neutral and acidic glycosphingolipids, quantitatively recovered and purified to homogeneity, were resolved by TLC. We used these techniques to determine sphingolipids and sphingoids of vertebrate central nervous system (CNS) tissue, using as little as 30-50 mg (wet weight) of tissue. In addition, phosphatidylcholine and sphingomyelin, relevant to ceramide metabolism, were quantitatively recovered in pure form and resolved by TLC. This method, used to study CNS sphingolipid content, may well be applicable to determine the sphingolipid composition of other tissues and cell culture, but further experiments are necessary to ascertain this.
Dasgupta
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Department of Neurology, Medical University of South Carolina, Charleston, SC 29425, USA. dasgupta@musc.edu
Hogan
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D000818:Animals; D002417:Cattle; D002490:Central Nervous System / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D013107:Sphingolipids / Q000032:analysis*
D013107:Sphingolipids