Introduction
Fatty liver disease results from the buildup of lipids, particularly triacylglycerols. The manifestation of the disease is influenced by the vagal nerve of the central nervous system. Our goal was to explore the lipid changes linked to vagal nerve activity in a clinical cohort, using a randomized modified sham feeding experiment with human participants. Triacylglycerols and phosphatidylcholines are the primary lipid species found in plasma and very low-density lipoproteins. To assess these well-known lipids, along with other potential markers, we conducted a targeted lipidomics assay using a Vanquish UHPLC system (Thermo Fisher Scientific) combined with an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Fisher Scientific).
Methods
The chromatographic separation of lipids was carried out on the UHPLC system equipped with a reversed phase based Accucore C18(2.6-μm,150×2mm) column. Mobile phase_A contained acetonitrile/water (50/50-vol/vol) solution and phase_B consisted of acetonitrile/isopropanol/water(10/88/2-vol/vol/vol), both phases containing 10mM ammonium formate and 10% formic acid. The flow rate was set to 400μL permin. A gradient of mobile phase_B was applied to ensure optimal separation of lipids. The MS scan range was set to 250–1200m/z for both +/- ionization mode. Fixed collision energy mode set to 35% and an inclusion list was used to obtain tandem MS spectra. Data analysis was performed with TraceFinder software. Labelled internal standards signal was used to obtain semi-quantitative values for lipids.
Preliminary Data
The targeted lipidomics assay covered 350 lipid species across 21 lipid classes, incorporating 13 labeled internal standards, including 15:0-18:1 (d7) PI, 15:0-18:1 (d7) PS, 15:0-18:1 (d7) DAG, 15:0-18:1 (d7) PC, 15:0-18:1 (d7) PE, 15:0-18:1 (d7) PG, 15:0-18:1(d7)-15:0 TAG, 18:1(d7) CE, 18:1(d7) LPC, 18:1(d7) LPE, 18:1(d9) SM, 18:1/18:0(d5) GlcCer, and Cholesterol(d7)(-H2O). The lipidome composition of VLDL particles and plasma was measured in 10 healthy male participants, each undergoing either water or modified-sham-feeding (MSF) with a 48-hour interval between the two conditions. In total, 76 samples were analyzed. For low-abundant lipid classes like LPE, DAG, Cer, PE-O, CE, PE, LPE, GluCer, and LacCer, the detection threshold was set to 150,000 peak area units, while for TAGs, PC, SM, PC-O, LPC, PS, PG, and PI, it ranged from 300,000 to 500,000. We detected 203 lipid species in plasma and 95 in VLDL. As anticipated, triacylglycerols and phosphatidylcholines were the predominant lipid classes. While biological variability is expected in randomized crossover experiments with human participants, variability was less pronounced in VLDL, highlighting the robustness of the lipidomics assay. Notably, certain tri- and di-acylglycerol species, as well as unsaturated TAGs (db = 2, db = 3, db = 4, db = 5), decreased 48 hours following MSF. The lipid composition in both VLDL and plasma remained stable after the MSF intervention. Future studies could expand on this work by exploring the brain-vagus-liver axis, particularly in patients with fatty liver disease.