Analysis of the Effects of Brefeldin A on Deuterium-Labeled Sphinganine Metabolism Using Liquid Chromatography-Tandem Mass Spectrometry.

Ceramide (Cer) is synthesized in the endoplasmic reticulum (ER) using sphinganine as the common backbone and is then transported to the Golgi apparatus to synthesize two complex sphingolipids, sphingomyelin (SM) and glucosylceramide (GlcCer). Brefeldin A (BFA) affects the structure of the Golgi apparatus, resulting in the redistribution of the Golgi proteins into the ER. Therefore, BFA has been used to examine the ER-to-Golgi trafficking of lipids, but the detailed lipid changes in cells upon BFA treatment are not fully understood. Here, we examined the metabolism of deuterium-labeled sphinganine in HEK293T cells treated with BFA using liquid chromatography-tandem mass spectrometry. The BFA-pretreated cells were incubated in the presence of deuterium-labeled sphinganine and BFA for 5 min to 8 h, and the levels of dihydroceramide, Cer, dihydrosphingomyelin, SM, GlcCer, and phosphatidylcholine (PC) were investigated. The levels of Cer species in BFA-treated cells were lower than those in untreated cells within 2 h of incubation, but following >4 h of incubation, the levels of C14-20 Cer, encompassing C14-20 acyl chains, were similar in BFA-treated and untreated cells. Furthermore, BFA treatment increased the levels of C14-20 GlcCer but had little effect on those of C22-24 GlcCer. Moreover, after incubation for >4 h, BFA treatment decreased the levels of saturated C30-32 PC but had almost little effect on those of PC containing a monounsaturated C32-34 acyl chain. Our findings showed that BFA affected the metabolism of various lipids depending on the length and saturation of the fatty acid chain.