Mark Wefers Bettink

Chapter 5 90 during and after application of pressure at an interval of 1Hz and using 1 laser pulse per measurement. We have described the fundamental principles behind the technology and have provided a working implementation of the technique for ODR measurements in vivo (Harms et al., 2013) and a method to calculate ODR from the mitoPO 2 kinetics. On each time point mitoPO 2 and ODR are presented as average of 4 replicate measurements. In the time control group at T1 a starting mitoPO 2 of around 50 mmHg was chosen, to match the LPS group, for fitting of the ODR. This was done to prevent potential negative effects of increased noise at higher mitoPO 2 readings on ODR. Mitochondrial assay in muscle biopsies At the end of the experiment a muscle biopsy was taken from the m. quadriceps femoris. The biopsy was transferred to mitochondrial respiration buffer (MRB, a hybrid buffer consisting of 110 mM sucrose, 60 mM potassium lactobionate, 20 mM taurine, 10 mM monobasic potassium phosphate, 3 mM magnesium chloride, 20 mM HEPES, 1 mM EGTA, and 0.1% (w/v) BSA at pH 7.1 at 37°C) on ice, homogenized using a Potter-Elvehjem PTFE pestle and glass tube, and injected in the respirometer. A small piece of muscle was snap-frozen in liquid nitrogen and stored at -80 C for later determination of complex concentration and activity. High-resolution respirometer Oxygen consumption was measured using a high-resolution respirometer (Oxygraph O2k, Oroboros, Innsbruck, Austria). Prior to homogenate loading the instrument was calibrated following the manufacturer instructions and loaded with 2.1 ml MRB. An oxygen solubility in water of 0.92 was used to calculate oxygen levels. 0.1 ml of muscle homogenate was added to the respirometer, and the chamber was closed. All chemicals for the respirometer experiments were obtained from Sigma-Aldrich (Darmstadt, Germany). To determine mitochondrial complex activity, first pyruvate (5mmol/l final concentration) and malate (2mmol/l final concentration) were added. Subsequent addition of ADP (0.25 mmol/l final concentration) showed full activity of complex 1. After addition of rotenone (0.5 micromol/l final concentration) to the chamber complex 1 activity was stopped and succinate (5 mmol/l final concentration) was added to measure complex 2 activity. The ATPase was inhibited by adding oligomycin (2.5 micromol/l final concentration) and subsequently FCCP (1 mmol/l solution) titration was performed to determine maximal oxygen consumption. The oxygen consumption measured by the high-resolution respirometer was corrected for citrate synthase activity, an indicator