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N-actylsphingosine (C2-ceramide)is a synthetic water-soluble ceramide mimicking the activity of natural ceramides.By fixing chiral conformation on carbon numbers 2 and 3 in the ceramide structure, four chiral C2-ceramides naming d-erythro-, l-erythro-, d-threo and l-threo C2-ceramide were synthesized. We have investigated the chiral effects of the of these C2-ceramides on the sphingolipid metabolism, particularly on both HL-60 and U937 cells, the chiral C2-ceramide(10mcM) showed sphingosine accumlation monitored fluoromatrically by a high performance liquid chromatographic separation of the sphingoid bases. Most importantly, in HL-6O cells, l-erythro C2-ceramide induced a 50 fold increase in sphingosine as compared to the control, while l-threo C2-ceramide exhibited a minimal 7-fold increase. In contrast, sphinganine, another sphingoid base, showed less accumulation by any chiral C2-ceramide tested under the same conditions. These results suggested that chiral C2-ceramide primarily acts on the sphingolopid degradation pathway rather than on the sphingolipid biosynthetic route. The strong G0/G1 phase arrest in the cell cycle by treatment of l-erythro C2-ceramide indicates that the blockade of the sphingolipid degradation pathway might be concomitantly involved in the dysfunction of the cell cycle. On the other hand, the fact that all chiral C2-ceramides may increase sphinglsine by activation various ceramidases by which natural ceramides are divided into sphingosine and free fatty acids. However, the precise steps involved in this interaction are still unknowns.
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