title: High-precision isotope ratio mass spectrometry and stable isotope precursors for tracer studies in cell culture.
abstract: The use of stable isotope-labeled tracers is demonstrated in an in vitro system with analysis by high-precision isotope ratio mass spectrometry (IRMS), using n-3 long-chain polyunsaturated fatty acid (LCP) biosynthesis from [U-(13)C]18:3n-3 (18:3n-3*) in Y79 human retinoblastoma cells as a model system. The cells were cultured as a suspension in RPMI 1640 medium supplemented with 15% fetal calf serum at 37 degrees C with 5% CO(2) in air. They were harvested by sedimentation and cell lipids were extracted to determine the presence of 18:3n-3* metabolites using gas chromatography-combustion (GCC)-IRMS. As the dose of 18:3n-3* was systematically increased from treatment to treatment, the atom percent excess and the amounts of biosynthesized LCP* increased, while the percentage dose in each n-3 LCP* remained constant. Cultures incubated with 0.5 micromol (10 microM) of albumin-bound 18:3n-3, composed of 18:3n-3* diluted 1/60 or 1/100 with natural abundance 18:3n-3, yielded products with enrichments about 1.5 at.% excess (delta(13)C(PDB) < 1500 per thousand), which is optimal for high-precision measurements. Kinetics in Y79 cells incubated with 18:3n-3* showed that n-3 LCP* incorporation increased over time; 18:3n-3*, 20:5n-3*, 22:5n-3*, and 22:6n-3* were detected at all time points with the 1/60 dilution. These data document experimental parameters for optimal stable isotope use and IRMS detection for in vitro tracer methodology.
authors.affiliation: Division of Nutritional Sciences, Cornell University, Ithaca, New York 14850, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Huang
authors.forename: S
authors.initials: S
authors.lastname: Muddana
authors.forename: E N
authors.initials: EN
authors.lastname: Horowitz
authors.forename: C C
authors.initials: CC
authors.lastname: McCormick
authors.forename: J P
authors.initials: JP
authors.lastname: Infante
authors.forename: J T
authors.initials: JT
authors.lastname: Brenna
mesh_terms: D002247:Carbon Isotopes; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008433:Mathematics; D008745:Methylation; D019572:Retinal Neoplasms / Q000378:metabolism*; D012175:Retinoblastoma / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D015525:Fatty Acids, Omega-3