Mark Wefers Bettink

Interim analysis; Feasibility study of measurement of mitochondrial function in patients with sepsis 7 131 biosynthetic pathway. PpIX is synthesized in the mitochondria and administration of 5-aminolevulinc acid (ALA) substantially enhances the PpIX concentration. We used 8 mg ALAcare® plasters (photonamic GmbH und Co. KG, Pinneberg, Germany) for PpIX enhancement. Photoexcitation of PpIX populates the first excited triplet state, and causes the emission of red delayed fluorescence. The delayed fluorescence lifetime is inversely related to the mitoPO 2 according to the Stern–Volmer equation. The background of the delayed fluorescence lifetime technique is extensively described elsewhere.(20–22) The light source and the detection system are the two core components of the COMET monitor (Photonic Healthcare, Utrecht, the Netherlands). A 515 nm pulsed laser, pulse duration 60ns, with a 10Hz repetition rate illuminates the intracellular accumulated PpIX. The fluorescent signal is projected on a gated red-sensitive photomultiplier tube. The light emitted by the sensor is divergent and safe for eyesight at any distance. A detailed description of the COMET measuring system can be found elsewhere. (23) Mitochondrial oxygen consumption (mitoVO 2 ) ismeasuredby pressure-inducedocclusion of the microcirculation thereby stopping local oxygen supply. The measurement probe was held above the ALA treated skin by hand. Occlusion of the microcirculation in the skin was achieved by manual firm pressure with the measurement probe. This simple procedure reliably created a measurable mitoVO 2 , due to cessation of the microvascular oxygen supply and ongoing cellular oxygen consumption. The mitochondrial oxygen concentration was measured before and during application of pressure at an interval of 1 Hz, using two laser pulses per measurement. The mitoVO 2 is analyzed using Michaelis- Menten kinetics. We previously described these principles in detail and provided a working implementation of the technique for mitoVO 2 measurements. Platelet Mitochondrial Oxygen Consumption Chemicals and Sample Preparation All used chemicals were purchased from Merck, at the time of the order still known as Sigma-Aldrich (St Louis, MO, USA). In ICU-admitted participants, blood samples of about 10 mL were taken directly after completion of the COMET measurement from an existing arterial or venous line in K 2 E (EDTA) 6.0 mL tubes (BD Vacutainer ® , Becton Dickinson, Plymouth, United Kingdom). In patients without an existing arterial or venous line, for example after being transferred to other departments, blood was drawn via venous puncture in K 2 E (EDTA) tubes right after the measurement. Platelet-rich plasma (PRP) was then prepared within 10 – 30 minutes after the blood sample was taken, by centrifugation