title: Intramyocellular diacylglycerol concentrations and [U-¹³C]palmitate isotopic enrichment measured by LC/MS/MS.
abstract: 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.
authors.affiliation: Department of Physiology, Medical University of Bialystok, Bialystok, Poland. Electronic address: blacha@umb.edu.pl.
authors.forename: Agnieszka U
authors.initials: AU
authors.lastname: Blachnio-Zabielska
authors.affiliation: Department of Physiology, Medical University of Bialystok, Bialystok, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Zabielski
authors.affiliation: Endocrine Research Unit, Mayo Clinic, Rochester, MN.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Jensen
mesh_terms: D000818:Animals; D002247:Carbon Isotopes / Q000493:pharmacokinetics / Q000494:pharmacology; D002853:Chromatography, Liquid; D004075:Diglycerides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D013058:Mass Spectrometry; D018482:Muscle, Skeletal / Q000378:metabolism*; D019308:Palmitic Acid / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D002247:Carbon Isotopes; D004075:Diglycerides; D004791:Enzyme Inhibitors; D019308:Palmitic Acid
keywords: Diacylglycerols measurement; liquid chromatography; mass spectrometry; skeletal muscle