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

Sergey Tumanov, Vinay Bulusu, Eyal Gottlieb, Jurre J. Kamphorst

Acetyl-CoA is a key metabolic intermediate with roles in the production of energy and biomass, as well as in metabolic regulation. It was recently found that acetate is crucial for maintaining acetyl-CoA production in hypoxic cancer cells. However, the availability of free acetate in the tumor environment and how much tumor cells consume remains unknown. Similarly, much is still to be learned about changes in the dynamics and distribution of acetylation in response to tumor-relevant conditions. The analysis of acetate is non-trivial, and to help address these topics, we developed a rapid and robust method for the analysis of both free and bound acetate in biological samples. We developed a sensitive and high-throughput method for the analysis of acetate based on alkylation to its propyl derivative and gas chromatography-mass spectrometry. The method facilitates simultaneous quantification of both (12)C- and (13)C-acetate, shows high reproducibility (< 10 % RSD), and has a wide linear range of quantification (2-2000 μM). We demonstrate the method's utility by measuring free acetate uptake by cultured cancer cells and by quantifying total acetylation (using hydrolysis) in separate cellular compartments. Additionally, we measure free acetate in tissues and bio-fluids and show that there are considerable differences in acetate concentrations between organs in vivo, providing insights into its complex systemic metabolism and availability for various types of tumors. Our approach for the quantification of acetate is straightforward to implement using widely available equipment and reagents, and will aid in in-depth investigation of various aspects of acetate metabolism. It is also readily adaptable to the analysis of formate and short-chain fatty acids, making it highly relevant to the cancer metabolism community.