Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl-coenzyme A in muscle by liquid chromatography/mass spectrometry.
Recent diabetes and obesity research has been focused on the role of intracellular lipids in insulin resistance. Fatty acyl-coenzyme A (CoA) esters play a central role in the trafficking of intracellular lipids, but there has not previously been a method with which to quantify their kinetics using tracer methodology. We have therefore developed a high-performance liquid chromatography (HPLC)-mass spectrometry method to simultaneously measure the (13)C stable isotopic enrichment of palmitoyl-acyl-CoA ester and the concentrations of five individual long-chain fatty acyl-CoA esters extracted from muscle tissue samples. The long-chain fatty acyl-CoA can be effectively extracted from frozen muscle tissue samples and baseline separated by a reverse-phase HPLC with the presence of a volatile reagent-triethylamine. Negative ion electrospray mass spectrometry with selected ion monitoring was used to analyze the fatty acyl-CoAs to achieve reliable quantification of their concentrations and (13)C isotopic enrichment. Applying this protocol to rabbit muscle samples demonstrates that it is a sensitive, accurate, and precise method for the quantification of long-chain fatty acyl-CoA concentrations and enrichment.
Sun
Dayong
D
Metabolism Unit, Shriners Burn Hospital, University of Texas Medical Branch, 815 Market Street, Galveston, TX 77550, USA.
Cree
Melanie G
MG
Wolfe
Robert R
RR
D000214:Acyl Coenzyme A / Q000032:analysis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes