A rapid method for quantifying free and bound acetate based on alkylation and GC-MS analysis

Area of Science:

• Metabolic biochemistry
• Cancer biology
• Analytical chemistry

Background:

• Acetyl-CoA is vital for cellular energy, biomass, and metabolic regulation.
• Acetate is crucial for acetyl-CoA production in hypoxic cancer cells.
• Acetate availability and acetylation dynamics in tumors are not well understood.

Purpose of the Study:

• To develop a rapid and robust method for analyzing free and bound acetate in biological samples.
• To investigate acetate availability and consumption in the tumor microenvironment.
• To explore acetylation changes under tumor-relevant conditions.

Main Methods:

• Developed a sensitive, high-throughput method using alkylation and gas chromatography-mass spectrometry.
• Simultaneously quantified (12)C- and (13)C-acetate with high reproducibility and a wide linear range.
• Applied the method to measure acetate uptake in cancer cells and total acetylation in cellular compartments.

Main Results:

• Demonstrated the method's utility in measuring free acetate uptake by cultured cancer cells.
• Quantified total acetylation in separate cellular compartments.
• Measured in vivo acetate concentrations in tissues and biofluids, revealing significant organ-specific differences.

Conclusions:

• The developed acetate quantification method is straightforward and uses widely available equipment.
• The method aids in investigating various aspects of acetate metabolism.
• The approach is adaptable for analyzing formate and short-chain fatty acids, relevant to cancer metabolism research.