Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using glucose

Abstract

13C based Isotopic tracers of the media components can be used to kinetically track their contribution in the cell systems. A tracer ([U-13C6] glucose) was used to monitor its contribution into the central carbon metabolic pathways of human lung carcinoma (A549) cells by Gas chromatography-mass spectrometry (GC-MS) based mass isotopomer analysis. Calculated average 13C of methanolic extracts (glycerol: 5.46{+/-}3.53 % and lactate: 74.4{+/-}2.65 %), protein acid hydrolysates (serine: 4.51{+/-}0.21 %, glycine: 2.44{+/-}0.31 %, alanine: 24.56{+/-}0.59 %, glutamate: 8.81{+/-}0.85 %, proline: 6.96{+/-}0.53 % and aspartate: 10.72{+/-}0.95 %) and the culture filtrate (glycerol: 43.14{+/-}1.45 % and lactate: 81.67{+/-}0.91 %), showed significant contribution of 13C glucose. We observed the Warburg effect with higher levels of 13C lactate in the culture filtrate. 13C glycerol levels in culture supernatant showed significant increase with time and amino acids of glucogenic origin also contributed cellular protein biomass. The workflow adopted in this study for 13C analysis could be useful for the metabolic phenotyping of other mammalian cell systems under normal and perturbed (cancer and infection) conditions.