The oxygen partial pressure was measured using carbon fiber electrodes (Carbostar-1, Kation Scientific, Minneapolis, MN). The tip of the electrode was coated with 5% Nafion (Sigma-Aldrich, St. Louis, MO) to increase oxygen specificity. The process consisted of three individual Nafion coats. The microelectrodes were polarized at −0.8 V relative to a silver-silver chloride reference electrode (Cypress Systems, Lawrence, KS). Oxygen measurements were performed after 3-hour fasting using the two-electrode system (working and reference electrode), and the current generated was measured with a potentiostat and electrometer (Keithley model 610C; Cleveland, OH). The microelectrodes are calibrated at 37°C with 0, 5, 10, and 21% O2 gases (Airgas, Los Angeles, CA), and tissues were superfused (0.1 ml/min) with physiological Krebs salt solution. The tissue was maintained at 35° to 37°C by the heated Krebs solution. The solution was spread on the tissue as a thin film, drained into a platter, and drawn off by suction. The solution was equilibrated with 95% N2 and 5% carbon dioxide, which maintained the superfusate at a pH of 7.4 and minimized oxygen delivery to the tissue from the atmosphere. Oxygen measurements were made by penetrating the tissue with the microelectrode tip. The reference electrode was placed in the bath, and the microelectrode was placed in a shielded holder and advanced toward the measurement site with a micromanipulator. A long-working distance ×10 Leitz objective was used to direct the electrode to the measurement site. Before measurements, the electrode tip was immersed in the supernatant suffusion solution and the current was registered. The supernatant suffusion solution was set as 0 mmHg reference point. Upon introduction into the tissue, the microelectrodes responded with a time constant that was estimated to be of the order of 10 s. A stable reading was obtained within 30 s, and upon reaching the current plateau value, the electrode was extracted from the tissue and the tip was maintained within the suffusing saline solution. The unit of tissue O2 tension was converted from mmHg to % later. Oxygen tension was measured at >30 different regions of each mouse liver. Appearance rate denotes chances for observing indicated O2 tension in a random spot of the mouse liver.

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