title: Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry.
abstract: This study describes the use of a stable-isotope labeled precursor ([U-¹³C]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 (¹³C 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-¹³C]palmitic acid, ∼60% of the total palmitoyl-CoA was ¹³C-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-sphingomyelins 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 ∼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.
authors.affiliation: Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Haynes
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Allegood
authors.forename: Elaine W
authors.initials: EW
authors.lastname: Wang
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Kelly
authors.forename: M Cameron
authors.initials: MC
authors.lastname: Sullards
authors.forename: Alfred H
authors.initials: AH
authors.lastname: Merrill
mesh_terms: D000215:Acylation; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010168:Palmitates / Q000737:chemistry / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000096:biosynthesis*; D013107:Sphingolipids / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D005227:Fatty Acids; D010168:Palmitates; D013107:Sphingolipids; D010171:Palmitoyl Coenzyme A