Anne-Marie Koop

4 183 again centrifuged at 7200 g for ten minutes at 4°C. The pellet was suspended in 200 μl of ice cold medium A and transferred into an eppendorf and kept on ice during the experiment. Mitochondrial respiration Mitochondrial respiration was measured in a stirred, 2-channel high-resolution respirometry (Oroboros, Innsbruck, Austra) in the isolated mitochondria. The oxygen consumption rate was measured using two both in the presence of ADP. First pyruvate respirationwas assessed by 2 mM pyruvate, the second protocol contained 25 mM Palmitoyl-CoA and 2 mM L-carnitine. 2 mMmalate was added to the medium (MIR05) as well. State 3 was reached by adding 10 mM glucose, 1.5 U/ml hexokinase and 1 mM ATP, state 4 by adding 1.25 µM carboxyatractyloside (CAT) and state 3 uncoupled by added 1.5 µM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Oxygen consumption ratewas corrected for protein content. Citrate synthase activity kit (Sigma Aldrich, USA) was used as a marker of mitochondrial density. Internal standards used for lipidomic analysis A defined amount of internal standards (0.5 nmol of DG(14:0)2, 0.5 nmol of TG(14:0)2, 0.5 nmol of CE(14:0), 1.0 nmol of CL(14:0)4, 0.02 nmol of BMP(14:0)2, 2.0 nmol of PC(14:0)2, 0.5 nmol of PG(14:0)2, 1.0 nmol of PS(14:0)2, 1.0 nmol of PE(14:0)2, 0.2 nmol of PA(14:0)2, 0.5 nmol of PI(8:0)2, 0.2 nmol of SM(14:0)2, 0.02 nmol of LPG(14:0), 0.1 nmol of LPE(14:0), 0.5 nmol of LPC(14:0), 0.05 nmol of LPA(14:0) (purchased from Avanti Polar Lipids, Alabaster, AL, USA)) was used.