Intramyocellular diacylglycerol concentrations and [U-¹³C] palmitate isotopic enrichment measured by LC/MS/MS

Diacylglycerols (DAG) are important lipid metabolites thought to induce muscle insulin resistance when present in excess; they can be synthesized de novo from plasma free fatty acids (FFA) or generated by hydrolysis of preexisting intracellular lipids. We present a new method to simultaneously measure intramyocellular concentrations of and the incorporation of [U- ¹³C]palmitate from an intravenous infusion into individual DAG species. DAG were extracted from pulverized muscle samples using isopropanol: water:ethyl acetate (35:5:60; v:v:v). Chromatographic separation was conducted on reverse-phase column in binary gradient using 1.5 mM ammonium formate, 0.1% formic acid in water as solvent A, and 2 mM ammonium formate, 0.15% formic acid in methanol as solvent B. We used UPLC-ESI⁺-MS/MS in the multiple reaction monitoring (MRM) mode to separate the ions of interest from sample. Because DAG are a neutral lipid class, they were monitored as an ammonium adduct [M+NH₄]⁺. To measure isotopic enrichment (for ¹³C16:0/16:0-DAG and ¹³C16:0/C18:1-DAG), we monitored the basic ions as [M+2+NH₄]⁺ and the enriched compounds as [M+16+NH₄]⁺. We were able to measure concentration and enrichment using 20 mg of skeletal muscle samples obtained from rats receiving a continuous infusion of [U-¹³C]palmitate. Applying this protocol to biological muscle samples proves that the method is sensitive, accurate, and efficient.