Mark Wefers Bettink

Monitoring mitochondrial respiration 5 91 of the number of mitochondria was measured according to Srere (1969) (Srere and Lowensteijn, 1969) of the homogenate to enable objective comparison between samples. Complex determination To obtain an indication of the function of the oxidative phosphorylation the activity of citrate synthase (CS) and the activity of complex 1 and 2 were analyzed. Activities of the complexes were based on the amount of CS to make objective comparison possible. Tissue homogenates were prepared from frozen muscle in 0.25 M sucrose, 10 mM N-[2- hydroxyethyl] piperazine-N′-[2 ethylsulfonic acid](HEPES) and 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), pH 7.4. Enzyme activities were measured in the homogenates. Activities of the complexes of the mitochondrial respiratory chain were determined in muscle homogenates by spectrophotometric methods as described before (Scholte et al., 1995) Complex I or NADH-Coenzyme Q reductase was determined kinetically by following the rotenon sensitive decrease in the amount of NADH. Complex II or Succinate Coenzyme Q reductase was measured by following the TTFA dependent reduction of DCPIP after addition of succinate. Citrate synthase was used as an indicator for the number of mitochondria to enable an objective comparison. Statistical analysis In experiment A data are expressed as median with interquartile range. For intragroup analysis the Wilcoxon matched pair signed rank test was used, for intergroup analysis the Mann-Whitney test was used. For experiment B data are expressed as means with standard deviation (SD), unless stated otherwise. A paired t-test was used to detect intragroup differences. Two-way ANOVA with repeated measurements, using post hoc multiple comparison with Bonferoni correction, was used to detect intergroup differences. For the hemodynamic data only intergroup differences were tested compared to time control. Normality was tested by Q-Q analysis and the Shapiro-Wilk test. A p-value < 0.05 was considered statistically significant. Statistical calculations were performed using Graphpad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA).