Synthesis of ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids from lignoceroyl-CoA by rat brain microsomes.

The conversion of [1-14C]lignoceroyl-CoA to nonhydroxy- and alpha-hydroxyceramides and cerebrosides by brain microsomes of developing rat in the presence of NADPH was investigated. A new technique of thin layer chromatography for the separation of these lipids and unreacted substrate was developed for this assay. The synthesis of nonhydroxy- and hydroxyceramides was significantly stimulated by the addition of heat-stable factor, a factor which is essential in the alpha-hydroxylation of free lignoceric acid (I. Singh and Y. Kishimoto, manuscript in preparation). The addition of sphingosine also stimulated the ceramide synthesis to a great extent. When the microsomes or heat-stable factor were crude, the ceramides formed were further converted to cerebrosides, apparently by UDP-galactose contamination. The purification of these subcellular components resulted in the formation of only ceramides which, in turn, were converted to cerebrosides by the addition of UDP-galactose. These observations indicate that hydroxyceramide is the precursor of hydroxycerebroside and is formed by alpha-hydroxylation of lignoceroyl-CoA and N-acylation of sphingosine. However, lignoceroyl-CoA, like free lignoceric acid, does not appear to be the immediate substrate of the alpha-hydroxylation.