Factors to consider in using [U-13C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry

This study describes the use of a stable-isotope labeled precursor ([U-C-13]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (C-13 appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-C-13]palmitic acid, similar to 60% of the total palmitoyl-CoA was C-13-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelin were 62 +/- 3, 13 +/- 2, and 60 +/- 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16: 0-ceramide using exponential growth modeling (119 +/- 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least similar to 1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.-Haynes, C. A., J. C. Allegood, E. W. Wang, S. L. Kelly, M. C. Sullards, and A. H. Merrill, Jr. Factors to consider in using [U-C-13] palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry. J. Lipid Res. 2011. 52: 1583-1594.