Introduction
Fatty liver disease is caused by the accumulation of lipids, especially triacylglycerols. Disease phenotypes are regulated by the vagal nerve of the central nervous system. We aimed to characterize the lipid changes associated with vagal innervation in a clinical cohort using a randomized modified sham feeding experiment with human subjects. The main lipid species known to occur in plasma and very low-density lipoproteins are Triacylglycerols and Phosphotidylcholines. To measure these known lipids and other potential markers, we performed a targeted lipidomics assay on a Lumos Fusion Orbitrap instrument operating in full MS scan mode in both ESI-positive and -negative ionization mode that offered us the highest sensitivity and resolution.
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
Targeted lipidomics assay comprised 350 lipid species, covering 21 lipid classes and 13 labelled internal standards (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, Cholesterol(d7)(-H2O)). We measured the lipidome composition of VLDL particles and plasma in 10 healthy male volunteers, each fed with either water or modified-sham-feeding and vicee-versa after 48 hrs. In total, 76 samples were measured. Lipid detection threshold was set to 150000 units (peak area) for low abundant lipid classes (LPE, DAG, Cer, PE-O, CE, PE, LPE, GluCer, LacCer) and 300000-500000 for TAGs, PC, SM, PC-O, LPC, PS, PG, PI. In total, we detected 203 and 95 lipid species in Plasma and VLDL respectively. Major lipid classes were as expected: Triacylglycerols and Phosphotidylcholines. Although biological variability in human subjects is expected to be higher in such a randomized crossover experiment design with human subjects, the observed variability was less prominent in VLDL, indicating robustness of the lipidomics assay. Certain tri- and di-acylglycerols species as well as unsaturated TAGs (db = 2, db = 3, db = 4 and db = 5) decreased 48 hours after MSF. Remaining lipidome composition remained constant in both VLDL and Plasma after MSF intervention. In future, our study focusing on brain-vagus-liver axis could be expanded to patients suffering from Fatty Liver Disease. Lipidomics data have been deposited to the EMBL-EBI MetaboLights database with the identifier MTBLS4231.