Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Abstract

Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13) C]palmitate and [U(13) C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.