Unmasking the Lipid Landscape: Carbamazepine's Impact on Leydig Cells

This study focuses on evaluating the effects of selected CBZ concentrations (25 or 200 uM) on the lipid homeostasis of BLTK-1 murine Leydig cells. By employing liquid chromatography-mass spectrometry (LC-MS). Lipids were separated by a C18 reverse phase column, using an Ascentis® Express 90 Å C18 HPLC column (15 cm x 2.1 mm; 2.7 μm, Supelco®) inserted into an HPLC system (Ultimate 3000 Dionex, Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled online to a Q-Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). A volume of 5 μL of each sample mixture was injected into the HPLC column, at a flow rate of 260 μL/min. The temperature of the column oven was maintained at 50 °C. Elution started with 32% of mobile phase B, and the gradient used was: 45% B (1.5 min), 52% B (4 min), 58% B (5 min), 68% B (8 min), 70% B (11 min), 85% B (14 min), 97% B (18 min, maintained for 7 min), and 32% B (25.01 min, followed by a re-equilibration period of 8 min prior next injection).The Q-Exactive™ orbitrap mass spectrometer with a heated electrospray ionization source was operated in the positive mode (electrospray voltage of 3.0 kV) and negative modes (electrospray voltage of −2.7 kV). The sheath gas flow was 35 U, the auxiliary gas was 3 U, the capillary temperature was 320 °C, the S-lenses RF was 50 U and the probe`s temperature was 300 °C. Full scans MS spectra were acquired both in positive and negative ionisation modes in an m/z range of 300-1600, with a resolution of 70.000, automatic gain control (AGC) target of 1x106 and maximum injection time of 100 ms. For tandem MS (MS/MS) experiments, a top-10 data-dependent method was used. The top 10 most abundant precursor ions in full MS were selected to be fragmented in the collision cell HCD. A stepped normalized collision energy™ scheme was used and ranged between 25 and 30 eV for positive ion mode and between 20, 24 and 28 for the negative ion mode. The MS/MS spectra obtained were those combining the information obtained with the different collision energies applied to each ionization mode. The MS/MS spectra were obtained with a resolution of 17,500; an AGC target of 1x105; an isolation window of 1 m/z; and a maximum injection time of 100 ms. The cycles consisted of one full scan mass spectrum and ten data-dependent MS/MS scans, which were repeated continuously throughout the experiments, with the dynamic exclusion of 30 s and an intensity threshold of 8x104. Data acquisition was carried out using the Xcalibur data system (V3.3, Thermo Fisher Scientific, USA). PC, LPC and SM were analysed in the LC-MS spectra in the positive ion mode, as [M+H]+ ions. The presence of the fragmention at m/z 184, corresponding to the phosphocholine polar head group, in the MS/MS of [M+H]+ ions allows identifying PL molecular species belonging to the PC, LPC and SM classes, which were further differentiated by the characteristic retention times. The identification of PC, LPC and SM classes was confirmed in the LC-MS spectra in the negative ion mode, as formate adducts ([M+HCOO]− ions). MS/MS spectra of [M+HCOO]− ions of these three PL classes should display the typical fragment ion at m/z 168 (phosphocholine polar head group minus a methyl moiety). Carboxylate anions of fatty acyl chains can also be seen for PC and LPC. PE and LPE classes were analysed in negative ion mode ([M−H]− ions). The fragment ion at m/z 140 (phosphoethanolamine polar head group) and the carboxylate anions of fatty acyl chains can be found in the MS/MS data from negative ion mode. PI and PS species were analysed in negative ion mode, as [M−H]− ions. The presence of the fragment ion at m/z 241, corresponding to the phosphoinositol polar head group, in the MS/MS of [M-H]- ions, allows the identification of PI molecular species. PS species were identified in the MS/MS of [M-H]- ions by the neutral loss of -87Da from the molecular ion. Diacylglycerols (DG) and triacylglycerols (TG) were identified by [M+NH4]+ ions.