Tracing of Amino Acids Dynamics in Cell Lines Based on ¹⁸O Stable Isotope Labeling

Metabolite levels and turnover rates are necessary to understand metabolomic dynamics in a living organism fully. Amino acids can play distinct roles in various cellular processes, and their abnormal levels are associated with pathological conditions, including cancer. Therefore, their levels, especially turnover rates, may provide enormous information about a phenotype. ¹³C- or ¹³C,¹⁵N-labeled amino acids have also been commonly used to trace amino acid metabolism. This study presented a new methodology based on ¹⁸O labeling for amino acids that relied on monitoring mass isotopologues to calculate the turnover rates of amino acids. The method optimization studies were carried over for selective amino acid monitoring. This methodology provides a rapid, robust, and simple GC-MS method for analyzing the fluxes of amino acid metabolism. The developed method was applied to fetal human colon (FHC) and human colon carcinoma (Caco-2) cell lines to determine cancer-induced shifts in the turnover rates of amino acids. These results defined metabolic reprogramming in Caco-2 cells through increased glutamate and serine turnovers and sharply decreased turnovers of aspartate, threonine, and methionine, therefore pointing to some metabolic vulnerabilities in the metabolism of cancerous cells. The simple mechanism of the developed methodology, the availability of affordable ¹⁸O-enriched water, and the ease of application can open a new arena in fluxomics analysis.