High-precision isotope ratio mass spectrometry and stable isotope precursors for tracer studies in cell culture

The use of stable isotope-labeled tracers is demonstrated in an in vitro sy stem with analysis by high-precision isotope ratio mass spectrometry (IRMS) , using n-3 long-chain polyunsaturated fatty acid (LCP) biosynthesis from [ U-C-13]18:3n-3 (18:3n-3*) in Y79 human retinoblastoma cells as a model syst em. The cells were cultured as a suspension in RPMI 1640 medium supplemente d with 15% fetal calf serum at 37 degreesC with 5% CO2 in air. They were ha rvested by sedimentation and cell lipids were extracted to determine the pr esence of 18:3n-3* metabolites using gas chromatography-combustion (GCC)-IR MS, As the dose of 18:3n-3'* was systematically increased from treatment-to treatment, the atom percent excess and the amounts of biosynthesized LCP*i ncreased, while the percentage dose in each n-3 LCP* remained constant. Cul tures incubated with 0.5 mu mol (10 muM) Of albumin-bound 18:3n-3, composed of 18:3n-3* diluted 1/60 or 1/100 with natural abundance 18:3n-3,yielded p roducts with enrichments about 1.5 at.% excess (delta C-13(PDB) < 1500<part s per thousand>), which is optimal for high-precision measurements. Kinetic s in Y79 cells incubated with 18:3n-3* showed that n-3 LCP* incorporation-i ncreased over time; 18:3n-3*, 20: 5n-3*, 22:5n-3*, and 22:6n-3* were detect ed at all time points with the 1/60 dilution, These data document experimen tal parameters for optimal stable isotope use and IRMS detection for in vit ro tracer methodology. (C) 2000 Academic Press.