FIGURE 1.

A, HPLC analysis of accumulated lipids in FB1-treated HEK^SPTLC3 cells. HEK^SPTLC3 cells were cultured in the presence of FB1 for 24 h. The accumulated sphingoid bases were extracted, derivatized with ortho-phthaldialdehyde, and analyzed on a C18 reverse phase column with fluorescence detection. In the presence of FB1 (black line) sphinganine accumulated (SA, retention time 13.5 min), and a second unknown metabolite appeared (retention time 8.2 min). This unknown metabolite was partially superimposed by the internal standard (C₁₇-SO) because of the similar retention times. No accumulation of these metabolites was observed when the cells were treated with the SPT inhibitor myriocin (gray line). B, MS analysis with a serially arranged MS detector revealed an m/z of 450.3 for the unknown metabolite. The single ion chromatogram showed a single peak (m/z 450.3, gray line) with a retention time identical to that observed in the fluorescence spectrum. The internal standard (C₁₇-SO, m/z 462.3, thin black line) eluted shortly before the unknown metabolite. C, SA and the unknown metabolite did not accumulate in FB1 + myriocin-treated HEK^SPTLC3 cells.