Mark Wefers Bettink

Interim analysis; Feasibility study of measurement of mitochondrial function in patients with sepsis 7 133 attributed to non-mitochondrial oxygen consumption (proteins and enzymes in the serum). As these measurements were performed in serum rather than buffer, oxygen consumption of other processes in the platelets as well as oxygen consumption of the serum are part of the measured respiration. To correct for these facts, the remaining oxygen respiration rate was subtracted from the measured unstimulated respiration rate, the ADP phosphorylation independent respiration state and themaximal respiration rate. Figure 1 shows a representative oxygen respiration curve measured with HRR. The final platelet concentration in the chamber was measured at the Department of Clinical Chemistry (AKC) using an automated hematology analyzer (XN-10, Sysmex ® , Kobe, Japan). The O 2 flux per volume was then corrected for platelet concentration. Figure 1. Respirometry graph of oxygen consumption in platelets. Oxygen concentration per volume [nmol/mL] (Y 1 ) and flux per volume [pmol/(s × ml)] (Y 2 ) over time. Additions: OM (oligomycin) 4 µL, FCCP (11 times) 1 µL, R (rotenone) 1 µL and AM (antimycin A) 2 µL. Mitochondrial DNA measurement The analysis is based on the method described by Nakahira et al.(8) A short description follows. DNA isolation from plasma For DNA isolation the DNeasy Blood & Tissue kit from Qiagen was used (#69504, Qiagen, Hilden, Germany). Prior to DNA isolation the plasma was thawed on ice. 100 µl of plasma was diluted with 100 µl PBS. The samples were mixed using a vortex and centrifuged at 700xg for 5 minutes at 4°C. 190 µl of the supernatant was transferred to a new