Quantitative assessment of brain microvascular and tissue oxygenation during cardiac arrest and resuscitation in pigs.

Cardiac arrest is associated with a very high rate of mortality, in part due to inadequate tissue perfusion during attempts at resuscitation. Parameters such as mean arterial pressure and end-tidal carbon dioxide may not accurately reflect adequacy of tissue perfusion during cardiac resuscitation. We hypothesised that quantitative measurements of tissue oxygen tension would more accurately reflect adequacy of tissue perfusion during experimental cardiac arrest. Using oxygen-dependent quenching of phosphorescence, we made measurements of oxygen in the microcirculation and in the interstitial space of the brain and muscle in a porcine model of ventricular fibrillation and cardiopulmonary resuscitation. Measurements were performed at baseline, during untreated ventricular fibrillation, during resuscitation and after return of spontaneous circulation. After achieving stable baseline brain tissue oxygen tension, as measured using an Oxyphor G4-based phosphorescent microsensor, ventricular fibrillation resulted in an immediate reduction in all measured parameters. During cardiopulmonary resuscitation, brain oxygen tension remained unchanged. After the return of spontaneous circulation, all measured parameters including brain oxygen tension recovered to baseline levels. Muscle tissue oxygen tension followed a similar trend as the brain, but with slower response times. We conclude that measurements of brain tissue oxygen tension, which more accurately reflect adequacy of tissue perfusion during cardiac arrest and resuscitation, may contribute to the development of new strategies to optimise perfusion during cardiac resuscitation and improve patient outcomes after cardiac arrest.

Early regional response of apoptotic activity in newborn piglet brain following hypoxia and ischemia.

Responses of selected neuroregulatory proteins that promote (Caspase 3 and Bax) or inhibit (Bcl-2, high Bcl-2/Bax ratio) apoptotic cell death were measured in the brain of piglets subjected to precisely controlled hypoxic and ischemic insults: 1 h hypoxia (decreasing FiO2 from 21 to 6%) or ischemia (ligation of carotid arteries and hemorrhage), followed by 0, 2 and 4 h recovery with 21% FiO2. Protein expression was measured in cortex, hippocampus and striatum by Western blot. There were no significant differences in expression of Caspase-3 between sham operated, hypoxic and ischemic groups. There were significant regional differences in expression of Bcl-2 and Bax in response to hypoxia and ischemia. The changes in Bcl-2/Bax ratio were similar for hypoxia and ischemia except for striatum at zero time recovery, with ischemia giving lower ratios than hypoxia. The Bcl-2/Bax ratio was also lower for the striatum than for the other regions of the brain, suggesting this region is the more susceptible to apoptotic injury.

Culture of a Planktonic Calanoid Copepod through Multiple Generations.

Acartia tonsa has been propagated through 12 filial generations during 1 year in small laboratory cultures. The mean generation time was 25 days at 17 degrees C on a slightly suboptimum, mixed algal diet. Body size, reproductive capacity, and generation time were unchanged during the culture period.

Production of carbon monoxide and gaseous hydrocarbons in seawater: relation to dissolved organic carbon.

Carbon monoxide, ethylene, and propylene were produced in illuminated, cell-free distilled water or natural seawater systems to which dissolved organic matter produced by phytoplankton had been added. Methane and the higher saturated gaseous hydrocarbons were not produced. In the dark, little or no carbon monoxide and no hydrocarbons were produced in the distilled water systems; only carbon monoxide was produced in natural seawater, but less was produced than in the light.

Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue.

The imaging of phosphorescence provides a method for monitoring oxygen distribution within the vascular system of intact tissues. Isolated rat lives were perfused through the portal vein with media containing palladium coproporphyrin, which phosphoresced and was used to image the liver at various perfusion rates. Because oxygen is a powerful quenching agent for phosphors, the transition from well-perfused liver to anoxia (no flow of oxygen) resulted in large increases of phosphorescence. During stepwise restoration of oxygen flow, the phosphorescence images showed marked heterogeneous patterns of tissue reoxygenation, which indicated that there were regional inequalities in oxygen delivery.

Carotid body sensory discharge and glomus cell HIF-1 alpha are regulated by a common oxygen sensor.

The carotid body responds to both acute and more prolonged periods of lowered oxygen pressure. In the acute response, the decrease in oxygen pressure is coupled to increased afferent neural activity while the latter involves, at least in part, increase in the hypoxia inducible transcription factor HIF-1 alpha. In this paper, we summarize evidence that both the acute changes in neural activity and the longer term adaptive changes linked to HIF-1 alpha induction share the same oxygen sensor, mitochondrial cytochrome c oxidase.

Trabecular anisotropy and collagen fibre orientation in the mandibular condyle following experimental functional appliance treatment using sheep.

In order to study the modifying effects of functional appliances on the mechanical environment of the temporomandibular joint (TMJ), we characterised the structure of the mandibular condyle subsequent to an experimental functional appliance intervention. Eight, four-month-old, castrated male Merino sheep, were randomly allocated to experimental and control groups (n = 4 in each group). Forward mandibular displacement was induced with an intraoral appliance. The study period was 15 weeks, during which time fluorochromes were administered to all of the animals. Midsagittal sections of the TMJ were selected for analysis and trabecular anisotropy was estimated using bone histomorphometry. Only the experimental group demonstrated that the trabecular bone in the central condylar region was less anisotropic when compared to the subchondral region. Also, the variation in trabecular anisotropy of the central condylar region was found to be smaller in the experimental group. The collagen fibre orientation was analysed under polarised light as the proportion of the dark or bright fibres observed in regions which existed before, and regions which formed during the experiment, as determined by the fluorochrome labels. In the experimental group, more bright collagen fibres were found in the most superior region of the mandibular condyle when compared with the controls. These results suggested that the experimental functional appliances changed the orientation and pattern of the mechanical forces acting on the mandibular condyle, and possibly increased the magnitude of the lateral functional forces applied to the most superior part of the condyle during such treatments.

Localization of tumors and evaluation of their state of oxygenation by phosphorescence imaging.

Oxygen-dependent quenching of phosphorescence has been used to image the distribution of oxygen pressure in small tumors and surrounding tissue. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected into the surface of the muscle of the hindquarter of rats, and the tumors were grown until they were 0.2-1.0 cm in diameter. The phosphorescent probe for oxygen was injected into the systemic blood, and phosphorescence was imaged with a video camera. Images of the phosphorescence were collected using a series of different delay times after illumination with a light flash (less than 5-microseconds width at half-height), and the phosphorescence decay constants (lifetimes) and oxygen pressure were calculated for each pixel of the image arrays. The areas of tissue within the tumors were observed to have increased phosphorescence lifetimes and lower oxygen pressures than the surrounding tissue. Phosphorescence imaging is, therefore, a noninvasive optical method which permits quantitation of the distribution of oxygen in small tumors and also, at least in the 9L tumors, differentiation of tumor from normal tissue.

Tamoxifen induces hypoxia in MCF-7 xenografts.

Tamoxifen is widely used as an adjunct therapy for breast cancer. We hypothesized that hypoxia develops in tumors as a result of tamoxifen treatment because tamoxifen has been reported to be antiangiogenic and thrombogenic. MCF-7 breast tumors were grown under estrogenic stimulation in 4-6-week-old CD-1 nu/nu female mice. When the tumors were approximately 5 mm in diameter, 17beta-estradiol pellets were replaced with either placebo or tamoxifen-containing pellets. Two days later, tissue oxygenation was measured using immunohistochemical detection of binding of the 2-nitroimidazole EF5. Intravascular oxygen partial pressures were measured noninvasively by oxygen-dependent quenching of phosphorescence of an injected dye that is excited by light pulses. Tamoxifen treatment increased hypoxia in the tumors, as measured by EF5 binding (P = 0.01 by Mann-Whitney test). This observation was not dependent on the presence of tamoxifen-induced necrosis. Intravascular oxygen partial pressures were lower in tumors relative to surrounding normal tissue in tamoxifen-treated tumors as compared to placebo-treated tumors. In vitro, tamoxifen did not modify the oxygen-dependent metabolism of EF5, indicating that the increased EF5 binding in tamoxifen-treated tumors reflects a physiological decrease in tissue oxygenation. The clinical significance of these observations is discussed in the context of the sequencing of tamoxifen with other therapies, and in light of recent data suggesting that hypoxia may be associated with genetic changes resulting in a more aggressive tumor phenotype.

Evaluation of enzyme systems and their regulation: the inapplicability of irreversible thermodynamics.

The formalism called irreversible thermodynamics has been examined for its applicability to the description of enzymic reactions. The basic assumption of this formalism is that the net flux through a reaction is related to the free energy change, a relationship which is usually assumed to be linear. These assumptions are shown to be approximately true for the trivial case of an enzyme reaction within 0.8 kJ/mol (0.2 kcal/mol) of equilibrium in the absence of changes in any regulatory parameters (such as inhibitor, activator, or enzyme concentration). For all other reaction conditions the net flux is not related to the free energy change and in special cases for which an apparent relationship is seen, it is not linear. Thus, application of the formalism of irreversible thermodynamics gives rise to qualitatively and quantitatively erroneous results and conclusions. Since most regulatory enzymic reactions are far from equilibrium, and the net reaction rate of such reactions is regulated by changes in inhibitors, activators and/or enzyme concentration, the formalism of irreversible thermodynamics is in general neither applicable nor useful in understanding the behavior of biological reaction systems.

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans.

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N3CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membrane of Paracoccus denitrificans and Tetrahymena pyriformis. The N3CCP uncouples respiration in P. denitrificans and T. pyriformis cells with U1/2 values of 1.05 microM and 0.24 microM, respectively. Binding studies show the presence of 0.65 +/- 0.05 high affinity sites per cytochrome alpha with Kd of 0.5 +/- 0.1 microM in P. denitrificans membranes and 1.4 +/- 0.2 sites per cytochrome alpha 2 with a Kd of 0.4 +/- 0.1 microM in T. pyriformis membranes. Irradiation of [3H]-N3CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10--15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647--656).

Coulometric and potentiometric evaluation of the redox components of cytochrome c oxidase in situ.

A new coulometric-potentiometric titration cuvette is described which permits accurate measurements of oxidation-reduction components in membranous systems. This cuvette has been utilized to measure the properties of cytochrome c oxidase in intact membranes of pigeon breast muscle mitochondria. The reducing equivalents accepted and donated by the portion of the respiratory chain with half-reduction potentials greater than 200 mV are equal to those required for the known components (cytochrome a3 and the high-potential copper plus cytochrome a, 'visible copper', cytochrome c1, cytochrome c, and the Rieske iron-sulfur protein). Titrations in the presence of CO show that formation of the reduced cytochrome a3-CO complex requires two reducing equivalents per cytochrome a3 (coulometric titration). Potentiometric titrations indicate (Lindsay, J.G., Owen, C.S. and Wilson, D.F. (1975) Arch. Biochem. Biophys. 169, 492--505) that both cytochromes a3 and the high-potential copper must be reduced in order to form the CO complex (n = 2.0 with a CO concentration-dependent half-reduction potential, Em). By contrast, titrations in the presence of azide show that the Em value of the high-potential copper is unchanged by the presence of azide and thus azide binds with nearly equal affinity whether the copper is reduced or oxidized.

Regulation of cellular energy metabolism: the Crabtree effect.

The Crabtree effect (inhibition of respiration by glycolysis) is observed in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Addition of glucose to aerobic suspensions of glucose-starved cells (Sarcoma 180 ascites tumor cells) causes a burst of respiration and lactate production due to ATP utilization for glucose phosphorylation by hexokinase and phosphofructokinase. This burst of activity is followed by inhibition of both respiration and glycolysis, the former to below the value before glucose addition (Crabtree effect). Both the respiratory rate and the glycolytic flux appear to be regulated by the cytosolic [ATP]/[ADP][Pi] albeit by completely different mechanisms. Respiration is regulated by the free energy of hydrolysis of ATP, such that the rate increases as the [ATP]/[ADP][Pi] decreases and decreases as the [ATP]/[adp][Pi] increases. The regulatory enzymes of glycolysis are activated by ADP (AMP) and Pi and inhibited by ATP. Thus both respiration and glycolysis increase or decrease as the [ATP]/[ADP][Pi] decreases or increases. The parallel regulation of both ATP-producing pathways by this common metabolite ratio is consistent with the cytoplasmic [ATP]/[ADP][Pi] being an important determinant of homeostatic regulation of cellular energy metabolism.

Studies of the orientation of the mitochondrial redox carriers. III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers.

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the gx and gy axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of gx and gy in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a3.

Studies on the orientations of the mitochondrial redox carriers. I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane.

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45 degrees between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2-1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a3. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90 degrees.

Studies on the orientations of the mitochondrial redox carriers. II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated, oriented mitochondrial multilayers.

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c1, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the "tightly bound" mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90 degrees.

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate.

The efflux of mitochondrial adenine nucleotide which is induced by addition of PPi to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 microM, Vmax being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The Km for PPi of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PPi.

Oxidation-reduction potentials of respiratory chain components in Thiobacillus A2.

(1) Cells of Thiobacillus A2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the 'invisible', or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.

The dental implications of bisphosphonates and bone disease.

In 2002/2003 a number of patients presented to the South Australian Oral and Maxillofacial Surgery Unit with unusual non-healing extraction wounds of the jaws. All were middle-aged to elderly, medically compromised and on bisphosphonates for bone pathology. Review of the literature showed similar cases being reported in the North American oral and maxillofacial surgery literature. This paper reviews the role of bisphosphonates in the management of bone disease. There were 2.3 million prescriptions for bisphosphonates in Australia in 2003. This group of drugs is very useful in controlling bone pain and preventing pathologic fractures. However, in a small number of patients on bisphosphonates, intractable, painful, non-healing exposed bone occurs following dental extractions or denture irritation. Affected patients are usually, but not always, over 55 years, medically compromised and on the potent nitrogen containing bisphosphonates pamidronate (Aredia/Pamisol), alendronate (Fosamax) and zolendronate (Zometa) for non-osteoporotic bone disease. Currently, there is no simple, effective treatment and the painful exposed bone may persist for years. The main complications are marked weight loss from difficulty in eating and severe jaw and neck infections. Possible preventive and therapeutic strategies are presented although at this time there is no evidence of their effectiveness. Dentists must ask about bisphosphonate usage for bone disease when recording medical histories and take appropriate actions to avoid the development of this debilitating condition in their patients.