Stable ¹³C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study

Area of Science:

• Cellular Metabolism
• Cancer Biology
• Analytical Chemistry

Background:

• Glutamine is a key nutrient in human blood, vital for nucleotide, lipid, and amino acid synthesis, and energy production.
• Studying glutamine metabolism, particularly lipid synthesis via reductive carboxylation, requires precise analytical methods.
• Glioblastoma (GBM) cells exhibit altered metabolic pathways crucial for their growth and survival.

Purpose of the Study:

• To present a detailed protocol for investigating glutamine metabolism in human glioblastoma (GBM) cells using stable isotope tracing.
• To enable the identification and quantification of polar metabolites and long-chain fatty acids (LCFAs) derived from 13C-glutamine.
• To distinguish between specific fatty acid isomers with identical masses and explore metabolic alterations in GBM cells.

Main Methods:

• Stable isotope tracing with 13C-glutamine in human glioblastoma cells.
• Untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS).
• Step-by-step protocol for metabolite extraction, detection, and isomer differentiation.

Main Results:

• The protocol successfully traces 13C-glutamine flux and identifies derived polar metabolites and LCFAs in GBM cells.
• The method distinguishes between palmitoleic acid (cis-9-hexadecenoic acid) and palmitelaidic acid (trans-9-hexadecenoic acid) isomers.
• Previously unknown metabolic alterations in GBM cells were identified following lysosome inhibition by pimozide.

Conclusions:

• This protocol provides a robust method for analyzing glutamine metabolism and its lipid derivatives in cancer cells.
• The technique is valuable for distinguishing between fatty acid isomers and understanding metabolic reprogramming.
• The findings offer insights into GBM cell metabolism and potential therapeutic targets, especially concerning lysosome function.