Mark Wefers Bettink

Chapter 4 66 Celsius. The oxygen concentration within the incubator was measured with a Fibox 4 trace (PreSense Precision Sensing GmbH, Regensburg, Germany). Prior to the mitoPO 2 measurements the arterial blood pressure was taken. The first mitoPO 2 measurement was done in the incubator at a low surrounding PO 2 achieved by filling the incubator with nitrogen gas (PO 2 < 5 mmHg), and with the blood pressure cuff pressurized to 50 mmHg above systolic pressure. After verification of the cessation of blood flow with an O2C laser-doppler monitor with an LFX-43 probe (oxygen to see version 2424, Lea Medizintechnik GmbH, Germany), local pressure was applied with the measurement probe of the COMET to empty and occlude the microvessels in the measured tissue in order to perform a dynamic measurement showing the decreasing mitoPO 2 (120 measurements at 1 Hz). With the combination of flow cessation with the pressure cuff, local pressure on the sensor, and mitochondrial oxygen consumption the minimal mitoPO 2 was determined in the arm during occlusion. The lowest measured mitoPO 2 during this measuring sequence was taken as lowest measurable mitoPO 2 per subject. An arterial blood sample was taken from the radial artery to determine arterial oxygen tension (PaO 2 ) with a blood gas analyzer (ABL 800 Flex, Radiometer, Brønshøj, Denmark). Nitrogen gas was mixed with room air to set the oxygen concentration in the incubator to a level equal to the arterial PaO 2 (range ±5 mmHg). In this study, topical application of cyanide was used to equalize mitoPO 2 to PaO 2 by blocking oxygen consumption at the skin measurement location. Cyanide ions (CN) bind with high affinity to the mitochondrial cytochrome c oxidase, blocking its activity. As a result, electron transport in the enzyme chain of oxidative phosphorylation and subsequently mitochondrial ATP production and mitochondrial oxygen metabolism are inhibited [16]. The applied cyanide cream was locally produced and contained a concentration of 1% potassium cyanide (Sigma-Aldrich, St. Louis, Missouri) mixed with hydrophilic cremor Lanette (Lanette cream I FNA, Bipharma, Weesp, The Netherlands). After 1 minute the cream was removed and the lower arm was placed in the air mixture equal to the sampled PaO 2 , after which the mitoPO 2 was measured. To test blockage of mitochondrial respiration after topical application of cyanide the absence of oxygen consumption was checked. After 20 measurements in a sequence of 120 measurements at 1 Hz pressure was applied to the COMET skin sensor. Without cyanide a decrease in mitoPO 2 within seconds was seen, as illustrated in figure 5a. While the mitochondrial respiration was blocked the mitoPO 2 remained constant as illustrated