High-Precision Isotope Ratio Mass Spectrometry and Stable Isotope Precursors for Tracer Studies in Cell Culture

✍ Scribed by Meng-Chuan Huang; Sri Muddana; Eric N. Horowitz; Charles C. McCormick; Juan P. Infante; J.Thomas Brenna

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 87 KB
• Volume: 287
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4843

✦ Synopsis

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.