C17 ceramide

10⁶ cells were subjected to lipid extraction with 1.5 mL methanol/chloroform (2:1, v:v)^{72 (link)}. The extraction solvent contained d₇-sphingosine (d₇-Sph), C17-ceramide, and C16-d₃₁-sphingomyelin (all from Avanti Polar Lipids) as internal standards. Sample analysis was carried out by liquid chromatography tandem-mass spectrometry (LC-MS/MS) using either a TQ 6490 mass spectrometer (for Sph) or a QTOF 6530 mass spectrometer (for ceramides and sphingomyelins) (both from Agilent Technologies, Waldbronn, Germany) operating in the positive electrospray ionization mode (ESI+). The following selected reaction monitoring (SRM) transitions were used for quantification: m/z 300.3 → 282.3 for Sph and m/z 307.3 → 289.3 for d₇-Sph. The precursor ions of ceramide or sphingomyelin species (differing in the lengths of their fatty acid chains) were cleaved into the fragment ions m/z 264.270 (for ceramide) or m/z 184.074 (for sphingomyelin), respectively^{73 (link)}. Quantification was performed with Mass Hunter Software (Agilent Technologies).

For lipid analysis 8 × 10⁶ PBL or 1 × 10⁶ BJAB cells per sample were resuspended in 500 μl methanol and subsequently subjected to lipid extraction using methanol/chloroform (2:1, v:v) (Gulbins et al., 2018 (link)). The extraction solvent contained d₇-Sph, d₇-sphingosine-1-phosphate (d₇-S1P), C17-ceramide, and C16-d₃₁-SM (all Avanti Polar Lipids, Alabaster, United States) as internal standards. Sample analysis was carried out by liquid chromatography tandem–mass spectrometry (LC–MS/MS) using either a TQ 6490 mass spectrometer (for Sph and S1P) or a QTOF 6530 mass spectrometer (for ceramides and SMs) (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+). The following selected reaction monitoring (SRM) transitions were used for quantification: m/z 300.3 → 282.3 for Sph, m/z 380.3 → 264.3 for S1P, m/z 307.3 → 289.3 for d₇-Sph, and m/z 387.3 → 271.3 for d₇-S1P. The precursor ions of ceramide or SM species (differing in their fatty acid chain lengths) were cleaved into the fragment ions m/z 264.270 or m/z 184.074, respectively (Kachler et al., 2017 (link)). Quantification was performed with Mass Hunter Software (Agilent Technologies).

Quadriceps muscles were homogenized in 14 µl/mg tissue of 3 : 4 water : methanol using a Precellys tissue homogenizer (setting: shaking at 6500 r.p.m. twice for 30 s). Chloroform containing 0.5 µM C17 ceramide (d18:1/17:0, Avanti Polar Lipids) was added to each sample at a ratio of 16 µl/mg tissue. The organic layer was transferred to a glass tube and dried under a gentle stream of nitrogen. Samples were reconstituted in isopropanol/methanol/chloroform (4/2/1) and 5 μl was injected on to a Waters ACQUITY ultra-performance liquid chromatography (UPLC) CSH C18 1.7 μm column (2.1 × 100 mm) maintained at 55 °C. The mobile phase, flow rate, and gradient conditions were as previously described^{69 (link)}. UPLC-QTOFMS analysis was performed in a Waters SYNAPT G2-Si in positive and negative electrospray ionization modes. Data were analyzed using Targetlynx XS and the percentage of neutral isotope was calculated as a peak area ratio of complete ¹³C-labeled metabolite/¹²C-labeled metabolite × 100%.

Diesel particulate extract used in the present study is Standard Reference Material 1975 (SRM 1975), purchased from the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). Nicotinamide, NADPH, NADP, and 2, 7-dichlorofluorescin diacetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Apo, N-acetylcystine. C17-S1P, S1P (d18:1), sphingosine, ceramides (fatty acid lengths C8, C12, C16, C18, C22, C24, and C24:1), C17-ceramide (d17:1/C18:0), and C12-sphingomyeline sphingosine, ceramides (fatty acid lengths C12, C16, C18, C22, C24, and C24:1) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Organic solvents for sphingolipid extraction or LC-MS/MS analysis were purchased from Merck (Darmstadt, Germany).

After treatment, HepG2 cell suspension and liver homogenate were collected. After protein measurement, the samples were fortified with 50 µL internal standard solutions and used for lipids extraction using 2 mL of extraction mixture (iso-propanol: water: ethyl acetate = 30:10:60; v: v: v). The samples were vortexed, sonicated 30 s for 3 times and centrifuged for 10 min at 4000 rpm. The supernatants were transferred into a new vial and re-extracted as described above. The extracted supernatants were dried under a gentle nitrogen stream and subjected to quantification of ceramides on an Agilent high performance liquid chromatography system coupled with a quadrupole-time of flight mass spectrometer (6545 Q-TOF), as described previously [27 (link),28 (link)], based on the C17-ceramide internal standards from Avanti Polar Lipids (Alabaster, AL, USA) as well as C14-ceramide, C18-ceramide, C20-ceramide, C22-ceramide, C24-ceramide, C24:1-ceramide and C26:1-ceramide standards from ZZStandard (Shanghai, China). Ceramide level is presented as µmol/protein (mg/mL).

Ceramides and sphingomyelin were extracted from spleen tissue and quantified as recently described [54 (link)]. Briefly, lipid extraction was performed using C17-ceramide and C16-d31-sphingomyelin as internal standards (Avanti Polar Lipids, Alabaster, AL, USA). Sample analysis was carried out by liquid chromatography tandem-mass spectrometry (LC-MS/MS) a Quadrupole-Time of Flight 6530 mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive ESI mode. The precursor ions of ceramides (C16-ceramide (m/z 520.508), C17-ceramide (m/z 534.524), C18-ceramide (m/z 548.540), C20-ceramide (m/z 576.571), C22-ceramide (m/z 604.602), C24-ceramide (m/z 632.634) and C24:1-ceramide (m/z 630.618)) were cleaved into the fragment ion m/z 264.270. The precursor ions of sphingomyelin (C16-sphingomyelin (m/z 703.575), C16-d₃₁-sphingomyelin (m/z 734.762), C18-sphingomyelin (m/z 731.606), C20-sphingomyelin (m/z 759.638), C22-sphingomyelin (m/z 787.669), C24-sphingomyelin (m/z 815.700) and C24:1-sphingomyelin (m/z 813.684)) were cleaved into the fragment ion m/z 184.074. Quantification was performed with MassHunter Software (Agilent Technologies, Santa Clara, CA, USA). Determined sphingolipid amounts were normalized to the protein content of the tissue homogenate used for lipid extraction.