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Wilson D.F.,University of Pennsylvania | Harrison D.K.,Microvascular Measurements
Advances in Experimental Medicine and Biology

Phosphorescence quenching and visible lightguide spectrophotometry were used to measure brain cortex oxygen partial pressure and skin oxygen saturation, respectively, during stepwise haemorrhage and re-transfusion in four 4-7-day-old anaesthetised piglets. In three cases, the effect of administration of adrenalin (epinephrine) was investigated. Brain cortex partial pressure was measured using a conventional phosphorescence pO2 probe (bc locpO2) and using a self-contained phosphorescence microsensor (bcmicropO2). Peripheral tissue oxygen saturation was measured on the skin of the abdomen (abSsO 2) and the distal right foreleg (flSsO2) using visible lightguide spectrophotometry. Haemorrhage of 65 ml reduced mean arterial blood pressure (MABP) from 75.5 ± 11.0 mmHg (mean ± standard deviation) to 42 ± 2.6 mmHg. Mean bclocpO2 fell from 30.1 ± 3.1 to 13.1 ± 2.5 mmHg and mean bc micropO2 fell from 33.8 ± 11.4 to 13.3 ± 9.5 mmHg. abSsO2 and flSsO2 values fell from 47.4 ± 8.1 % and 43.6 ± 10.9 %, respectively, to 21.9 ± 5.5 % and 23.8 ± 14.0 %. Infusion of adrenalin produced a mean transient increase in MABP to 137 ± 2.6 mmHg, falling to 75.7 ± 16.3 mmHg within 3 min. bclocpO2 also increased to 24.1 ± 14.6 mmHg, but there were no significant changes in bc micropO, abSsO2 or flSsO 2. Following reinfusion all parameters returned to values that were not statistically different from their pre-haemorrhage values. The dynamic recordings of all the oxygenation parameters indicated that they were sensitive indicators of the degree of haemorrhage during the experiments. © 2013 Springer Science+Business Media New York. Source

Wilson D.F.,University of Pennsylvania | Harrison D.K.,Microvascular Measurements | Vinogradov A.,Moscow State University
Journal of Applied Physiology

Cytochrome c oxidase is the enzyme responsible for oxygen consumption by mitochondrial oxidative phosphorylation and coupling site 3 of oxidative phosphorylation. In this role it determines the cellular rate of ATP synthesis by oxidative phosphorylation and is the key to understanding how energy metabolism is regulated. Four electrons are required for the reduction of oxygen to water, and these are provided by the one-electron donor, cytochrome c. The rate of oxygen consumption (ATP synthesis) is dependent on the fraction of cytochrome c reduced (fred), oxygen pressure (pO2), energy state ([ATP]/[ADP][Pi]), and pH. In coupled mitochondria (high energy state) and pO2 >60 torr, the rate increases in an exponential-like fashion with increasing fred. When the dependence on fred is fitted to the equation rate = a(fred)b, a decreased from 100 to near 20, and b increased from 1.3 to 4 as the pH of the medium increased from 6.5 to 8.3. During oxygen depletion from the medium fred progressively increases and the rate of respiration decreases. The respiratory rate falls to ? (P50) by about 1.5 torr, at which point fred is substantially increased. The metabolically relevant dependence on pO2 is obtained by correcting for the increase in fred , in which case the P50 is 12 torr. Adding an uncoupler of oxidative phosphorylation eliminates the dependence of the cytochrome c oxidase activity on pH and energy state. The respiratory rate becomes proportional to fred and the P50 decreases to less than 1 torr. © 2014 the American Physiological Society. Source

Harrison D.K.,Microvascular Measurements | Vaupel P.,TU Munich
Advances in Experimental Medicine and Biology

Heterogeneity is a feature of both normal oxygen supply to tissue and of a supply that is disturbed due to a wide range of different pathologies. Here, the physiological importance of heterogeneity of tissue oxygenation is revisited. The anatomical and functional basis for heterogeneity of blood flow, local and regional regulatory mechanisms in normal tissues and the pathophysiology of the failure of regulation will be examined. Under physiological conditions, regulation of blood flow distributions at global, regional and microregional levels play coordinated roles in ensuring adequate O2supply to all tissue cells. How this is achieved may be organ-/organ layer-specific, depending on its function and priorities to match local O2delivery to consumption. Examples where these regulatory mechanisms break down under conditions of ischaemia and shock will also be given. In contrast, pathologic heterogeneity in tissue oxygenation resulting from uncontrolled, chaotic growth as seen in malignant tumours represents a pathophysiological status that is not predictable which, in general, is associated with chronic and acute hypoxia. This can have fatal consequences due to hypoxia- induced (mal-)adaptive processes, malignant tumour progression and treatment resistance. © Springer Science+Business Media, LLC 2014. Source

Wilson D.F.,University of Pennsylvania | Harrison D.K.,Microvascular Measurements | Vinogradov S.A.,University of Pennsylvania
Journal of Applied Physiology

The oxygen dependence of mitochondrial oxidative phosphorylation was measured in suspensions of isolated rat liver mitochondria using recently developed methods for measuring oxygen and cytochrome c reduction. Cytochrome-c oxidase (energy conservation site 3) activity of the mitochondrial respiratory chain was measured using an artificial electron donor (N,N,N',N'-tetramethyl-p- phenylenediamine) and ascorbate to directly reduce the cytochrome c, bypassing sites 1 and 2. For mitochondrial suspensions with added ATP, metabolic conditions approximating those in intact cells and decreasing oxygen pressure both increased reduction of cytochrome c and decreased respiratory rate. The kinetic parameters [KM and maximal rate (VM)] for oxygen were determined from the respiratory rates calculated for 100% reduction of cytochrome c. At 22°C, the KM for oxygen is near 3 Torr (5 μM), 12 Torr (22 μM), and 18 Torr (32 μM) at pH 6.9, 7.4, and 7.9, respectively, and VM corresponds to a turnover number for cytochrome c at 100% reduction of near 80/s and is independent of pH. Uncoupling oxidative phosphorylation increased the respiratory rate at saturating oxygen pressures by twofold and decreased the KM for oxygen to <2 Torr at all tested pH values. Mitochondrial oxidative phosphorylation is an important oxygen sensor for regulation of metabolism, nutrient delivery to tissues, and cardiopulmonary function. The decrease in KM for oxygen with acidification of the cellular environment impacts many tissue functions and may give transformed cells a significant survival advantage over normal cells at low-pH, oxygen-limited environment in growing tumors. © 2012 the American Physiological Society. Source

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