IN RECENT years a number of investigators have reported the occurrence in mammalian tissue of long-chain bases other than sphingosine. Dihydrosphingosine was isolated from cerebrosides of beef brain and spinal cord by CARTER, HAINES, LEDYARD, and NORRIS (1947), from cerebrosides of human brain by SCHWARZ, DREISBACH, BARRIONUEVO, KLESCHICK and KOSTYK (1961), and from blood plasma by SWEELEY and MOSCATELLI (1959). The 20-carbon homologue of sphingosine was detected in sphingolipids of horse and beef brain by PROSTENIK and MAJHOER-ORESCANIN (1960), in bovine gangIiosides by KLENK and GIELEN (1961), in bovine mucolipids by STANACEV and CHARGAFF (1962), and in gangliosides but not in cerebrosides or sphingomyelins of several species by SAMBASIVARAO and MCCLUER (1 964). Very recently ROSENBERG and STERN (1966) reported that bases obtained from gangliosides of foetal rat brain were almost exclusively of the CIS type. During the post natal period, C,,-sphingosine increased to such an extent that both types of bases were about equal in the 2-5-weekold rat. They further noticed that the bases of cerebrosides and sphingomyelins conversely remained of the C,, type throughout all stages of brain development. This report describes the isolation of C,,-sphingosine and C,,-dihydrosphingosine from brain and spinal cord of normal and E.A.E. rabbits.
MATERIAL A N D METHODSDisease-free female albino rabbits of weighing 2.5-3 kg wt. from Huntingdon Farms, Philadelphia were divided into two groups. One group was used to produce E.A.E. by the method of HOYER, CONDIE and GOOD (1960), the other as controls. Shortly after sacrifice of the paralytic (E.A.E.) animals and controls, brains and spinal cords were excised and treated with chloroform-methanol (2: 1, v/v). The mixtures of extracted lipids were washed according to FOLCH, ASCOLI, LEES, MEATH and LEBARON (1951) and separated on two columns (ROUSER, BAUMAN and KRITCHEVSKY, 1961) for isolation of cerebrosides and sphingomyelins.Isolation of cerebrosides and sphingornyelins. The isolation of the sphingolipids was carried out essentially in the following manner. The washed lipid extract containing 70-100 mg of cerebrosides, 200-300 mg of phospholipids, and other lipids was evaporated to dryness at 35" in a nitrogen stream. The residue dissolved in a small amount of chloroform-methanol (7 : 1, v/v) was applied to a column of 50 g DEAE-cellulose. Approximately 500 ml of that solvent were passed through the column at the rate of 1 ml/min until the eluate emerging from the column showed a positive ninhydrin reaction according to ROUSER et al. (1961). The ninhydrin negative fraction was evaporated to dryness with a stream of nitrogen, re-dissolved in a small amount of chloroform, and applied to a hydrated silicic acid silicate coIumn (ROUSER et al.
