Application Note 31
Tracing Lipid Diposition in vivo Using Stable Isotope-Labeled Fatty Acids and Mass Spectrometry
David G. McLaren, Steven J. Stout, Sheng-Ping Wang, Paul L. Miller, Kithsiri Herath, Vinit Shah, Ablatt Mahsut, Jose Castro-Perez, Douglas G. Johns, Michele Cleary, Stephen F. Previs, and Thomas P. Roddy
Merck Research Laboratories, Merck & Co., Inc. Rahway, NJ 07065-0900 USA
Lipids are ubiquitous molecules which serve a variety of important biological functions, including energy storage (triglycerides), modulation of cellular membrane structure and function (phospholipids and cholesterol), intracellular signaling, and hormonal regulation. Dysfunctions of lipid metabolism contribute to a variety of diseases including, among others, atherosclerosis, hypertriglyceridemia, and type 2 diabetes. As such, understanding the synthesis, regulation, and transport of lipids in the body is important to developing new and improved therapies for these diseases. Stable isotopes have been used to study several aspects of lipid metabolism including the synthesis and disposition of cholesterol,1,2 phospholipids,3 and VLDL triglycerides.4 In this application note, we highlight some of the advantages and experimental considerations for using stable isotope-labeled fatty acids as substrates to study lipid metabolism in vivo in mice.
Experimental Design
For experiments on lipid synthesis, C57BL6 mice were treated with a vehicle control or a systemic, small-molecule inhibitor of microsomal triglyceride transfer protein (MTP).5 One hour later, the mice were administered 150 mg/kg of oleic acid, potassium salt (13C18, 98%) CP 95% (CLM-8856) mixed with corn oil (Figure 2) or 20% TPGS (Figure 3). Blood samples were withdrawn at serial timepoints following tracer administration and processed to plasma. 10 µL of plasma were mixed with 90 µL of methanol containing heavy internal standards and further diluted with 300 µL of pentanol. The samples were centrifuged briefly to pellet insoluble proteins and 5-10 µL of the supernatant were analyzed by ultraperformance liquid chromatography interfaced with either a triple quadrupole (Waters Xevo TQ) or quadrupole time-of-flight (Waters Synapt G2) mass spectrometer. For experiments on fatty acid oxidation, mice were administered a cocktail containing 150 mg/kg of three fatty acids – palmitic acid, stearic acid, and oleic acid mixed with intralipid. Experiments were conducted in which one of the fatty acids was perdeuterated palmitic acid (D31, 98%) (DLM-215), stearic acid (D35, 98%) (DLM-379), oleic acid (D33, 98%) (DLM-1891), and the remaining two were unlabeled, or in which all three fatty acids were perdeuterated. 10 µL of plasma were incubated with acetone at basic pH to exchange deuteriums in the plasma water with acetone. The deuterium enrichment in acetone was then measured by headspace gas chromatography interfaced with an isotope ratio mass spectrometer (Thermo Scientific). Further details on the methods employed in these experiments can be found in references.6-8
Data Analysis
Total plasma triglycerides were analyzed using a commercially available biochemical kit (Thermo Scientific). All other data were acquired and analyzed using the Waters or Thermo instrument software packages. Multiple-reaction monitoring on a triple quadrupole mass spectrometer was used to trace the appearance of oleic acid, potassium salt (13C18, 98%) in selected triglycerides and cholesteryl ester in the blood. The concentration of 13C18-oleate-labeled lipids was determined from the peak area ratio of the analyte to its matched, heavy-labeled internal standard. Concentrations were plotted as a function of time, and the area under the curve was determined using the Graphpad Prism software package. Alternatively, data were manually mined from full-scan MSe spectra obtained on the quadrupole time-of-flight instrument. Selected ion chromatograms were extracted, and the relative abundance of each 13C18-oleate-labeled lipid was determined based on the peak area ratio to a class-specific internal standard.
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