Abstract
The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoA concentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoA levels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extraction followed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoA in individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoA levels.
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Acknowledgements
We thank Drs. Kamelia Behnia, Qi Wang, and Mr. James Smalley for compound A PK exposure work; Drs. Simeon Taylor, Adrienne Tymiak, and Mary Ann Pelleymounter for support; Drs. Mark Sanders and David Wang-Iverson for many stimulating discussions and Ms. Mary Ellen Salyan for some of the initial investigations.
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Joelle M. Onorato and Luping Chen contributed equally to this work.
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Onorato, J.M., Chen, L., Shipkova, P. et al. Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue. Anal Bioanal Chem 397, 3137–3142 (2010). https://doi.org/10.1007/s00216-010-3879-1
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DOI: https://doi.org/10.1007/s00216-010-3879-1