Preparation of 13C-labeled ceramide by acetic acid bacteria and its incorporation in mice

We prepared 2-hydroxypalmitoyl-sphinganine (dihydroceramide) labeled with a stable isotope by culturing acetic acid bacteria with C-13-labeled acetic acid. The GC/ MS spectrum of the trimethylsilyl derivative of C-13-labeled dihydroceramide gave molecular ions with an increased mass of 12-17 Da over that of nonlabeled dihydroceramide. The fragment ions derived from both sphinganine base and 2-hydroxypalmitate were confirmed to be labeled with the stable isotope in the spectrum. Therefore, C-13-labeled dihydroceramide can be an extremely useful tool for analyzing sphingolipid metabolism. The purified [C-13] dihydroceramide was administered orally to mice for 12 days, and the total sphingoid base fractions in various tissues were analyzed by GC/MS. The spectrum patterns specific to C-13-labeled sphingoids were detected in the tissues tested. Sphinganine pools in skin epidermis, liver, skeletal muscle, and synapse membrane in brain were replaced by [C-13] sphinganine at about 4.5, 4.0, 1.0, and 0.3%, respectively. Moreover, about 1.0% of the sphingosine pool in the liver was replaced by [C-13] sphingosine, implying that exogenous dihydroceramide can be converted to sphingosine. These results clearly indicate that ingested dihydroceramide can be incorporated into various tissues, including brain, and metabolized to other sphingolipids.-Fukami, H., H. Tachimoto, M. Kishi, T. Kaga, H. Waki, M. Iwamoto, and Y. Tanaka. Preparation of C-13-labeled ceramide by acetic acid bacteria and its incorporation in mice. J. Lipid Res. 2010. 51: 3389-3395.