In Vivo Studies on the Introduction of the 4-t-Double Bond of the Sphingenine Moiety of Rat Brain Ceramides

Erythro - dl -[3- 3 H]sphinganine was injected directly into the brains of 14- to 15-day-old rats. After 2 hours, 2.0% of the injected radioactivity could be recovered in the ceramide fraction of the brain lipid extract. The ceramide material was fractionated by thin layer chromatography into the classes of N -acylsphinganine and N -acylsphingenine, containing approximately equal amounts of radioactivity. Free 3 H-labeled long chain bases were recovered after alkaline hydrolysis of the two ceramide classes and confirmed to be sphinganine and sphingenine. The presence of tritium in the unsaturated base sphingenine indicates a direct conversion of sphinganine to sphingenine or an acylated sphinganine to acylated sphingenine. No free [ 3 H]sphingenine could be demonstrated in the lipid extract indicating either that it is present in very small amounts, due perhaps to rapid acylation, or that it is not produced as the free base but rather through acylated sphinganine. A time study over 145 min of the incorporation of [3- 3 H]sphinganine into ceramides showed an initial rapid production of N -acyl-[ 3 H]sphinganine, which stayed at a steady level for the duration, while N -acyl-[ 3 H]sphingenine appeared more slowly but was the predimonate ceramide at the final set time. This suggests N -acylsphinganine may be the precursor of N -acylsphingenine. When N -[1- 14 C]stearoyl-[3- 3 H]sphinganine, with an isotope ratio of 2.22 ( 3 H: 14 C), was the injected substrate, ceramide containing [ 3 H]sphingenine and 14 C fatty acid could be isolated. The observed isotone ratio for the N -acylsphingenine was 2.52, indicating an 87% retention of 14 C compared to 3 H. The similarity of ratios suggests a direct conversion of the injected ceramide to the isolated compound; the small loss of 14 C due perhaps to some hydrolysis followed by resynthesis.