Novel fluorescent oxygen indicator for intracellular oxygen measurements

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. In this paper, we describe the application of the oxygen sensing indicator, ruthenium dibipyridine 4-(1"-pyrenyl)-2,2'-bipyridine chloride [Ru(bpy-pyr)(bpy)(2)], for molecular oxygen measurement in J774 murine macrophages. Ru(bpy-pyr)(bpy)(2) exhibits strong visible absorption, efficient fluorescence, long excited state lifetime, large Stokes shift, and high photo- and chemical stability. The fluorescence of Ru(bpy-pyr)(bpy)2 is efficiently quenched by molecular oxygen. It is 13 fold higher in a nitrogenated solution than in an oxygenated one. The dye passively permeates into cells and maintains its oxygen sensitivity for at least 5 h when the cells are stored in a phosphate buffered saline solution at pH 7.4. The oxygen sensitivity, photostability, and chemical stability of the indicator and the effect of hypoxia and hyperoxia on the intracellular oxygen level in single macrophages are discussed.     

Measurement of oxygen tension by the oxygen cathode

The usefulness of the flush-ended electrode forin vivo measurements of oxygen tension in tissue has been restricted by the lack of a satisfactory theoretical or empirical relationship between the polarographic current and the oxygen tension in the tissue. It is shown here that, if the electrode is small enough (so that its oxygen consumption is negligible compared with that of the nearby tissue), the current is proportional to the mean value over the electrode surface of the values that the oxygen tension would have taken if the potential had not been applied. The proportionality factor depends on the capillary-tissue structure in the neighbourhood of the probe. However, a membrane covering the surface of the electrode can be constructed so as to eliminate this effect and so enable comparisons between the levels of oxygen tension to be made at different points in the tissue. At the same time, the loss of sensitivity to variations of oxygen tension in time can be reduced to negligible proportions. The current, in this case, is proportional to the mean undisturbed value of the oxygen tension over the surface of the membrane.     

Time dependence of singlet oxygen luminescence provides an indication of oxygen concentration during oxygen consumption

Singlet oxygen plays a major role in photodynamic inactivation of tumor cells or bacteria. Its efficacy depends critically on the oxygen concentration [O(2)], which can decrease in case oxygen is consumed caused by oxidative reactions. When detecting singlet oxygen directly by its luminescence at 1270 nm, the course of the luminescence signal is critically affected by [O(2)]. Thus, it should be feasible to monitor oxygen consumption during photo-oxidative processes. Singlet oxygen was generated by exciting a photosensitizer (TMPyP) in aqueous solution (H(2)O or D(2)O) of albumin. Chromatography shows that most of the TMPyP molecules are unbound, and therefore singlet oxygen molecules can diffuse in the solution. A sensor device for oxygen concentration revealed a rapid decrease of [O(2)] (oxygen depletion) in the solution during irradiation. The extent of oxygen depletion in aqueous albumin solution depends on the radiant exposure and the solvent. When detecting the luminescence signal of singlet oxygen, the shape of the luminescence signal significantly changed with irradiation time. Thus, local oxygen consumption could be monitored during photodynamic action by evaluating the course of singlet oxygen luminescence.     

Systemic oxygen delivery by peritoneal perfusion of oxygen microbubbles

Severe hypoxemia refractory to pulmonary mechanical ventilation remains life-threatening in critically ill patients. Peritoneal ventilation has long been desired for extrapulmonary oxygenation owing to easy access of the peritoneal cavity for catheterization and the relative safety compared to an extracorporeal circuit. Unfortunately, prior attempts involving direct oxygen ventilation or aqueous perfusates of fluorocarbons or hemoglobin carriers have failed, leading many researchers to abandon the method. We attribute these prior failures to limited mass transfer of oxygen to the peritoneum and have designed an oxygen formulation that overcomes this limitation. Using phospholipid-coated oxygen microbubbles (OMBs), we demonstrate 100% survival for rats experiencing acute lung trauma to at least 2 h. In contrast, all untreated rats and rats treated with peritoneal oxygenated saline died within 30 min. For rats treated with OMBs, hemoglobin saturation and heart rate were at normal levels over the 2-h timeframe. Peritoneal oxygenation with OMBs was therefore shown to be safe and effective, and the method requires less equipment and technical expertise than initiating and maintaining an extracorporeal circuit. Further translation of peritoneal oxygenation with OMBs may provide therapy for acute respiratory distress syndrome arising from trauma, sepsis, pneumonia, aspiration, burns and other pulmonary diseases.     

On the pulmonary toxicity of oxygen: III. The induction of oxygen dependency by oxygen use

Oxygen is central to the development of neonatal lung injury. The increase in oxygen exposure of the neonatal lung during the onset of extrauterine air breathing is an order of magnitude, from a range of 10-12 to 110-120 Torr. The contributions of oxygen and the volume and pressure relationships of ventilatory support to lung injury are not easily distinguished in the clinical setting. Sequential changes in inspired air or 100% oxygen were studied in 536 newborn rabbits without ventilatory support. Bilateral cervical vagotomies (BCV) were performed at 24 h post natal to induce ventilatory distress which eventuates in hyaline membrane disease. The sequences applied yielded evidence for an induced state of oxygen dependency from oxygen use which was reflected in differences in survival and the extent of pulmonary injury. The median survival for animals kept in air throughout was 3 h. Oxygen before vagotomy or during the first 3 h afterwards extended the survival significantly but produced more extensive, more severe, and more rapid lung lesions. Returning animals to air after prior oxygen exposure reduced the number of survivors past 10 h and shortened the maximum survival in those groups. These features indicate the development of a dependency of the defense mechanisms on the availability of oxygen at the higher level for metabolic and possibly other aspects of the pulmonary and systemic response to injury, beyond the usual physiological need. Subset analysis revealed additive and latent effects of oxygen and demonstrated a remarkable rapidity in onset of severe lesions under some circumstances, illustrating the toxicity of oxygen per se.     

Perfluorocarbon-based oxygen delivery

The basic properties of perfluorocarbons (PFCs) and PFC emulsions relevant to their use as oxygen delivery systems are briefly reviewed. The key issues related to the selection of an appropriate, readily excretable PFC and the engineering of a stable injectable PFC emulsion are discussed. Oxygent, a terminally heat-sterilized, injectable 60% w/v PFC emulsion made primarily of F-octyl bromide and a few percent of F-decyl bromide, with egg phospholipids as an emulsifier, has been developed. Its efficacy in avoiding and reducing red cell transfusion during surgery has been established during a Phase III clinical evaluation. Another Phase III clinical trial in cardiopulmonary bypass surgery, with a protocol that included both augmented-acute normovolemic hemodilution and intraoperative autologous donation, has, however, been interrupted following the observation of adverse events. Data analysis assigned these events to an inappropriate study protocol. A search for possible interactions between Oxygent and fluids present during cardiopulmonary bypass surgery detected no effect of the emulsion on hemostasis, hemolysis and blood rheology.     

HIV: reactive oxygen species, enveloped viruses and hyperbaric oxygen

This paper demonstrates that there are many examples in the literature of contradictory data concerning reactive oxygen intermediates (ROIs), responsible for producing cellular oxidative stress (OS), and their enhancement or diminution of viral replication. Nevertheless, ROIs repeatedly have been shown to be virucidal against enveloped-viruses, like the human immunodeficiency virus (HIV). Hyperbaric oxygen therapy (HBOT) increases the production of ROIs throughout the body, leaving no safe harbor for the virus to hide outside the genome. This technique already has been tried on acquired immune deficiency syndrome (AIDS) patients, with exciting results. Historically, the biggest setback to demonstrating HBO's antiviral effects has been the investigator's folly of studying non-enveloped viruses or failing to initiate ROI production. ROIs specifically attack areas of unsaturation occurring in the polyunsaturated fatty acids of cell membranes and viral envelopes. Moreover, it consistently has been shown that a peroxidized viral envelope breaches, and a breached viral envelope causes viral disintegration.