Mark Wefers Bettink

Verification of calibration in man and comparison with vascular occlusion tests in healthy volunteers with COMET 4 65 fluorescence [1, 12]. To this end we cutaneously applied a 4 cm 2 plaster, containing 8mg ALA (Photonamic, Hamburg, Germany). Six to eight hours previous to the measurements the ALA-plaster was applied to the lower arm. The COMET Skin Sensor was fixated onto the skin using a double-sided adhesive transparent plaster without optical interference (LEA Medizintechnik GmbH, Giessen, Germany). 2.2 Verification of COMET calibration Since no gold standard exists to which COMET can be compared, verification of COMET calibration had to rely on creating predictable mitochondrial oxygen levels. We chose a two-point verification aiming at approximating zero oxygen conditions and arterial oxygen tension. In earlier experiments in cells and animals the oxygen tension was decreased by flushing or breathing nitrogen to wash out all available oxygen. In healthy human volunteers tissue-deoxygenation with nitrogen to a near-zero level is not a safe and viable option. A method that is applicable in humans is temporal arterial occlusion of a limb in combination with local pressure on the measuring probe. Arterial and microvascular occlusion inhibits blood flow and thus the oxygen supply to the measurement site. Ongoing cellular oxygen consumption will decrease local mitochondrial oxygen tension to very low values, approximating the desired zero oxygen conditions. In addition to measurements near zero oxygen conditions we applied a method to compare mitoPO 2 to arterial oxygen tension in a blood gas sample, in order to create a second calibration point at a higher PO 2 level. A known highmitochondrial oxygen tension can be achieved by abolishing the oxygen gradient between arterial blood and the tissue cells. After cessation of mitochondrial oxygen consumption diffusion equilibrates the mitochondrial and arterial oxygen tension. Mitochondrial respiration can be temporarily inhibited by locally applying cyanide [13–15], which has previously been demonstrated in cells and animals [8]. In the transient absence of mitochondrial oxygen metabolism, the measured mitoPO 2 can be compared to the oxygen partial pressure measured in an arterial blood gas (ABG) sample [13–15]. To diminish the influence of external factors like temperature and atmospheric oxygen a gas-sealed incubator was used to control internal air temperature and oxygen concentration, as shown in Figure 1a. During the measurements the subject’s arm was inserted into the incubator, which was set to an internal temperature of 37 degrees