Title: 4-O -methylhonokiol protects from alcohol/carbon tetrachloride-induced liver injury in mice

Transcription

1 Electronic supplementary material (ESM) J Mol Med 2017 Title: 4-O -methylhonokiol protects from alcohol/carbon tetrachloride-induced liver injury in mice Eleonora Patsenker 1,2, Andrea Chicca 3, Vanessa Petrucci 3, Sheida Moghadamrad 2, Andrea de Gottardi 2, 4, Jochen Hampe 5, Jürg Gertsch 3, Nasser Semmo 2,4*, Felix Stickel 1* 1 Division of Gastroenterology and Hepatology, University Hospital Zurich, Switzerland; 2 Department of Clinical Research, Hepatology, University of Bern, Switzerland; 3 Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland; 4 Department of Visceral Surgery and Medicine, Hepatology, Inselspital, University Hospital of Bern, Switzerland; 5 Medical Department 1, University Hospital Dresden, Technical University of Dresden, Germany. * The authors share equal contribution Corresponding author: Eleonora Patsenker eleonora.patsenker@usz.ch Tel. (+41)

2 Supplementary materials Table S1. List of antibodies and conditions used for Western Blotting Primary antibody GAPDH -tubulin SMA DGAT-2 p47-phox Anticytochrome p450 2E1 Bak Bax Bcl-xl Mcl-1 JNK1 CYP4A14 ALDH1A1 FAS Catalog number sc BT7R ab5694 sc sc ab28146 sc-832 sc S sc S sc PA sc Source goat Molecular Weight Dilution 37kDa 1:200 55kDa 1:2000 Incubation time Company 42kDa 1:200 RT, 2h abcam 44kDa 1:200 47kDa 1: kDa 1: kDa 1:400 23kDa 1:100 30kDa 1: , 40kDa 1:200 46, 54kDa 1: kDa 1:200 55kDa 1: kDa 1:200 ThermoFischer Scientific abcam Cell Signaling Cell Signaling ThermoFischer Scientific Secondary antibody goat anti goat anti donkey antigoat Catalog number sc-2005 sc-2004 sc-2020 Source Dilution Incubation time goat 1:2000 RT, 40 min goat 1:2000 RT, 40 min donkey 1:2000 RT, 40 min Company

3 Analytical Quantification of Endocannabinoid and Related Lipid Levels by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) (a) Chemicals and Reagents: All chemicals and reagents were of the purest analytical grade. AEA or (5Z,8Z,11Z,14Z)- eicosatetraenamide), AEA-d4 or N-(2-hydroxyethyl-1,1,2,2-d4)-(5Z,8Z,11Z,14Z)- eicosatetraenamide, 2-AG or (5Z,8Z,11Z,14Z)-eicosatetraenoic acid, 2-glyceryl ester, 2-AG-d5 or (5Z,8Z,11Z,14Z)- eicosatetraenoic acid, 2-glyceryl-1,1,2,3,3-d5 ester, almitoylethanolamide (PEA) or N-(2-hydroxyethyl)-hexadecanamide, PEA-d5 or N-(2-hydroxyethyl)- hexadecanamide-15,15,16,16,16-d4, or N-(2-hydroxyethyl)-octadecanamide, oleoylethanlonamide (OEA) or N-(2-hydroxyethyl)-9Z-octadecenamide, OEA-d4 or N-(2- hydroxyethyl-1,1,2,2-d4)-9z-octadecenamide, linoleoylethanolamide (LEA) or N-(2- hydroxyethyl)-9z,12z-octadecadienamide, LEA-d4 or N-(2-hydroxyethyl-1,1,2,2-d4)-9Z,12Zoctadecadienamide, arachidonic acid (AA) or (5Z,8Z,11Z,14Z)-eicosatetraenoic acid and AAd8 or (5Z,8Z,11Z,14Z)-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid, were purchased from Cayman Chemical Europe, Tallinn, Estonia. HPLC-grade methanol (CH3OH), HPLC-grade acetonitrile, ammonium acetate and formic acid were obtained from Sigma-Aldrich, Steinheim, Germany. HPLC-grade ethyl acetate and hexane were obtained from Acros Organics, New Jersey, USA. Deionized water (18.2 MΩ cm) was obtained from an ELGA Purelab Ultra Genetic system (VWS (UK) Ltd, ELGA LabWater, UK). (b) Quantification of endocannabinoids and other lipid by LC-MS/MS: Sample extraction Liver tissues were weighted (~40mg) when still frozen and homogenized using a BeadBeater (Mini-BeadBeater-24, BioSpec, Oklahoma, USA) as previously showed (Chicca et al in press). The homogenized tissue was rapidly transferred to glass tubes containing 1.5 ml of ethyl acetate/hexane (9:1) 0.1% formic and a mixture of ISs (internal standards), strongly vortex for

4 30 seconds and sonicated in cold bath for 5 min. Samples were centrifuged at 3000 rpm for 10 min at 4 C and kept for 1h at -20 C to freeze the aqueous phase. The upper organic phase was recovered in plastic tubes, evaporated and the extracts reconstituted in 50 µl of ACN/EtOH (8:2). 10 µl of the solution were injected in the LC-MS/MS system. LC-MS/MS conditions A hybrid triple quadrupole 4000 QTRAP mass spectrometer (AB Sciex Concord, Ontario, Canada) was used with a Shimadzu UFLC (Shimadzu Corporation, Kyoto, Japan) with cooled auto-sampler. Sample temperature was maintained at 4 C in the auto-sampler prior to analysis. The LC columns were Reprosil-PUR C18 column (3 μm particle size; 2 50 mm, Dr. A. Maisch, High Performance LC-GmbH, Ammerbuch, Germany) maintained at 40 C. The system was operated in positive and negative mode with 2 different elution mobile phases and gradient method. In positive mode the elution mobile phases were: water, 2 mm ammonium acetate, 0.1% formic acid (solvent A) and methanol, 2 mm ammonium acetate (solvent B). The gradient started at 15% B, increasing linearly to 70% at 3.5 min, then to 99% at 8 min, maintained until 12 min, with subsequent re-equilibration at 15% for further 2.5 min. The flow rate was 0.35mL/min. In negative mode the organic mobile phase (B) was substituted with acetonitrile 0.1% formic acid. The gradient started at 5% B, increasing linearly to 40% at 3 min, then to 65% at 9 min and linearly again to 95% at 10min; this was maintained until 14 min, with subsequent re-equilibration at 5% for further 3 min. The flow rate was 0.3mL/min. The following Multiple Reaction Monitoring (MRM) transitions were monitored for quantification of the analytes and for the internal standard: Positive mode: 2AG m/z , (IS: 2-AG-d ); AEA m/z , (IS: AEA-d ); LEA m/z , (IS: LEA-d ); OEA m/z , (IS: OEA-d ), PEA m/z , (IS: PEA-d ). Negative mode: AA m/z , (IS: AA-d ). Triplicate 11 points calibrations were prepared in

5 0.1 % formic acid plus 1% BSA, and the appropriate matrix. The concentration of calibrators moved from 20 ng/ml to ng/ml for AA, 0.2 ng/ml to 500 ng/ml for LEA, PEA, OEA, 0.08 ng/ml to 200 ng/ml for AEA, 2 ng/ml to 5000 ng/ml for 2-AG. To ensure that were constant background concentration of all endogenous analytes, bulk tissues were homogenized in 0.1% formic acid and used for validation and calibration experiments (the background was subtracted to spiked concentration levels). The slope, intercept, and regression coefficient of those calibration lines were determined. LC-MS/MS validation To determine intraday (n=6) and interday (n=6 over a period of 8 separated days) precision and accuracy, analytes were spiked into liver tissue at 3 concentrations representing the entire range of concentration (LQC, low quality control, MQC medium quality control, HQC high quality control) and homogenized with extraction solvent. Precision was calculated from the relative standard deviation of the replicates (RSD), also known as coefficient of variation (CV), and accuracy was calculated by comparison of measured levels of spiked analytes with expected concentrations. A CV values of 20% was considered to be acceptable for accuracy and precision at the lower quality control. For the analytes already present in blank matrix itself evaluation of matrix effect and recovery was performed with the deuterated analogue. Matrix factor values were all closed to 1 (no matrix effects) except for LEA, PEA, OEA which values varied between (ion suppression) indicating a significant matrix ionization effects in positive mode effecting the ethanolamides. Reference to supplementary material: Chicca А, Gachet МS, Petrucci V, Schuehly W, Charles R, Gertsch J (2015) 4 -Omethylhonokiol increases levels of 2-arachidonoyl glycerol in brain via selective inhibition of its COX-2-mediated oxygenation. J Neuroinflammation 12: 89