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

Abstract

Background: 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.

Measurement of cations, anions, and acetate in serum, urine, cerebrospinal fluid, and tissue by ion chromatography

“…Syringes and filters were flushed at least 39 with ultra-pure water prior to sample filtration. This diligence reduced acetate contamination in blank samples (negative control) to undetectable levels, which have not been previously reported in other methodologies for acetate measurements (Richards et al 1975;Tumanov et al 2016).…”

“…; Tumanov et al. ), is that there is heavy acetate contamination in water supplies, and even ultra‐pure water (>18 MΩ), overtime begins drawing in organics that contaminate the water used for sample preparation. We therefore obtained fresh ultra‐pure water (>18 MΩ) prior to sample extractions or sample preparation.…”

“…(Tumanov et al. ) Any amino acids, carboxylic acids or nucleophiles remaining in samples are capable of reacting with derivatizing reagents and forming side products, drastically reducing the apparent concentration of SCFA and or leading to wide ranges for reported values. BDMPH, (benzyldimethylphenylammonium hydroxide; MCF, methylchloroformate.…”

“…(Richards et al 1975) Reaction 2 shows acetate derivatization to propyl acetate using methylchloroformate as a derivatizing agent, followed by a subsequent reaction with propyl alcohol. (Tumanov et al 2016) Any amino acids, carboxylic acids or nucleophiles remaining in samples are capable of reacting with derivatizing reagents and forming side products, drastically reducing the apparent concentration of SCFA and or leading to wide ranges for reported values. BDMPH, (benzyldimethylphenylammonium hydroxide; MCF, methylchloroformate.…”

“…For small organic ions such as acetate, the typical method is that of an ester derivatization (Perry et al 2016;Richards et al 1975;Tumanov et al 2016;Turnbaugh et al 2006). This method requires an ester derivatizing agent, strong acids/bases and organic extractions followed by analysis on gas chromatography.…”