Abstract
Through a systematic examination of basic (cationic) lipids separated on Folch's lower phase from extracts of human brain by cation exchange chromatography on carboxymethyl Sephadex in a chloroform/methanol mixture, followed by successive chromatographies on Florisil and Iatrobeads columns, five compounds of basic lipids were separated. Two major unknown compounds A and B and a minor unknown compound C were separated, in addition to minor compounds sphingosine and N,N-dimethylsphingosine. This paper describes the isolation and chemical characterization of major unknown compounds A and B, which were found only in the white matter but not in the gray matter of the human brain. Unmodified psychosine (galactosylsphingosine) was essentially undetectable under the experimental conditions. Unknown compounds A and B were identified as novel plasmal (fatty aldehyde) conjugates of psychosine with cyclic acetal linkage at the galactosyl residue of psychosine. Fatty aldehydes were identified as mainly palmital (16:0) and stearal (18:0). Sphingosine was identified as d18:1 sphingosine. Faster migrating compound A had 3,4-cyclic acetal linkage, and slower migrating compound B had 4,6-cyclic acetal linkage (where m is 14 or 16 and n is 12) as shown below. [formula: see text] Preliminary studies showed that compounds A, B, and C had a weak inhibitory effect on protein kinase C (PKC) and had no cytotoxic effect. In contrast, psychosine displayed a strong cytotoxicity and inhibitory effect on PKC. Therefore, the process controlling the addition or deletion of plasmal cyclic linkage to psychosine could be a crucial step in regulation of PKC, src, or other kinases susceptible to psychosine.
Highlights
Through a systematic examinationof basic effect on PKC
B were identified as novel plasmal src and ras kinases acting on substrate’ bvuitrtuisally absent conjugates of psychosine with cyclic acetal linkage at the galactosyl residue of psychosine
Unknown compounds A, B, and C could be degraded to the same position as psychosine after weak acid hydrolysis catalyzed by HgC12in 0.1 N HCl (Feulgen's condition) (11)or 0.3 acetylated de-0-acetylated lipids were analyzed by +FAB-MS using
Summary
To the dried lower phase of brain extract, solvent A was added until the solution became clearly soluble. The combined hexane extracts were evaporated under N, stream a t 37 "C to -10 pl, diluted with hexane for analysis by GC-MS as described below Under these conditions, production of EME derivatives was favored over conversion to long chain dimethylacetals. Long Chain Aldehyde Analysis separated on TLC (see Fig. L 4 , lane 5 ) as described in the Fig. 1 Lipid samples (400-500 pg) were methanolyzed in 2.0 ml of 0.5 N legend. Inseparatetests, this concentration of triethylamine HCl, 5 M Hz0 in MeOH for 5.5 h a t 80 "C. To detect unknown compounds A, B, C with 0.5% orcinol (Sigma) in 10% sulfuric acid followed by heating in an oven at 120 "C
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