Electrode measurement of oxygen tension with 1-ms time resolution.

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neurtralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation o of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.     

An extremely rapid burst of respiration upon aeration of mitochondria: measurement of oxygen tension with 10-msec time resolution

A continuous-flow apparatus was devised which was capable of measuring oxygen tension with a time resolution of better than 10 msec. Measurements were made with this device on suspensions of beef heart mitochondria. Anaerobic suspensions of mitochondria were aerated, and oxygen tension versus time was measured for times of 15 msec and longer. During the first few hundred milliseconds after aeration, respiration proceeded at a rate much faster than had ever been observed by conventional measurements. After this period, the respiration subsided to a rate comparable to that usually observed. This burst of respiration occurred only under conditions which cyclically discharged the energized state. Resting mitochondria showed no such burst, but mitochondria did show a burst when the energized state was being utilized by proton ejection, cation accumulation, oxidative phosphorylation, or uncoupler action. In the presence of uncoupler, antimycin prevented any burst of respiration, but rotenone plus malonate did not. This last result indicated that the substrates utilized during the burst were the components of the electron-transfer chain which lie between the antimycin block and the rotenone and malonate blocks, components such as CoQ and nonheme iron. This burst of respiration has a time course similar to that of the respiration-dependent proton ejection by mitochondria.     

Oxygen tension measurement by a method of time selection using the static platinum electrode with alternating potential

1. The possibility of obtaining sustained and reproducible results in the analysis of dissolved oxygen with simple platinum electrodes by means of the application of a periodic potential pattern was explored over a wide range of frequencies and with a variety of wave forms. 2. Satisfactory results were obtained by the application in the frequency range of 5 to 10 C.P.M. of a square wave consisting of a positive and a negative pulse with interposed shorting periods and observing the current flowing at the end of each successive negative pulse. This was found to be linearly proportional to O(2) concentration for a pulse duration of the order of 1 second when the RC constant of the circuit was sufficiently small. 3. An instrument was developed to provide the required wave form and record the terminal currents of the negative pulses. The instrument provides either for recording of current voltage curves (polarograms) or for continuous recording at a fixed voltage of diffusion limited current values. 4. Typical measurements of oxygen uptake with yeast suspensions illustrate the application of the technique to problems requiring frequent determinations during short intervals. 5. Applications of this technique to biological and other problems are indicated with its limitations.     

Determination of non-uniform pulmonary ventilation by transcutaneous measurement of the oxygen tension in the arterial blood under an oxygen load

A method for determination of uneven ventilation in the lungs by the transcutaneous measurement of oxygen tension in arterial blood (TcPO2) is suggested. The degree of uneven ventilation was estimated by the time of a decrease in the TcPO2 till point of departure (desaturation time) after oxygen inhalation. The TcPO2 was measured by an oxymonitor (Hellige, Model SM 361). Twelve normal controls and 24 patients with chronic obstructive lung diseases were examined. The results showed that desaturation time in controls was 3.4 +/- 0.14 min, and in patients with chronic obstructive lung diseases it was significantly prolonged till 8 +/- 0.75 min; those data were in a good correlation with helium dilution time. The method may be recommended as a diagnostic test for determination of uneven ventilation in the lungs.     

Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats.     

The time for oxygen release in photosynthesis: reconciliation of flash polarography with other measurement techniques

The time for oxygen release in photosynthesis has been reported to be 30–130 ms when measured by flash polarography under low polarization voltages (Plijter et al. 1988), in opposition to 1–3 ms with light modulated oxygen polarography (Jolio et al. 1966), with the detection of produced oxygen in a flowing sample (Etienne 1968) or with photoacoustic detection of oxygen evolution (Canaani et al. 1988). However, we show here that flash polarographic measurements require properly cleaned electrodes, a precise polarization voltage, as well as a short polarization time of the electrodes. When these criteria were met, an oxygen release in less than 2 ms could be measured by flash polarography under low polarization voltages, in accordance with the other techniques. But under high polarization voltages, the interpretation of the polarographic response to oxygen production must take into account the diffusion of oxygen, the capacitance of the platinum electrode and the oxygen release time. We present a model of the electrode response taking into account these factors; by interpreting the response of the electrodes with this model, we found an oxygen release time of 1.7 ms. These evidences support strongly a short oxygen release time of 1–3 ms.     

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.     

Electron crystallography of bacteriorhodopsin with millisecond time resolution

The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin.     

Transcutaneous oxygen tension during exercise in patients with claudication.

Transcutaneous oxygen tension (TcPO2) was monitored during maximal exercise in 10 patients with stable moderate to severe claudication. The TcPO2 fell by 16% at the onset of claudication and 32% at the maximum walking distance. On resting this decrease reached a maximum of 66% roughly four minutes after exercise. This was followed by a steady recovery. The percentage changes were reproducible in each patient and were appreciably different from the TcPO2 exercise profiles of normal healthy volunteers. TcPO2 monitoring during exercise is a simple, reproducible, cheap, and useful technique for assessing claudication and compares favourably with other techniques used to quantify this condition.     

Cultivation of three-dimensional cartilage-carrier-constructs under reduced oxygen tension

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Experiments with different oxygen concentrations (21, 10 and 5%, v/v O(2)) were performed and the constructs were compared qualitatively and quantitatively. The appearance of the cartilage obtained under reduced oxygen tension seemed to be closer to native cartilage with respect to shape of the cells, distribution of the cells within the matrix, smoothness of the surface, etc. The thickness of the cartilage formed by free swelling was always in the same range as for native cartilage (approximately 1mm). Qualitatively the most stable attachment of the cartilage on top of the carrier was found for 10% O(2) (v/v). Especially at 5% O(2) (v/v) the attachment between cartilage and carrier was not sufficient. The constructs generated at lower oxygen tensions had a significantly higher amount of glycosaminoglycan per DNA, but still significantly less when compared to native cartilage. Furthermore, the cultivated cartilage contained a large amount of collagen type II. The experiments proved the applied concept for generation of cartilage-carrier-constructs and the usefulness of cultivation under reduced oxygen tension.     

Calculations of intracapillary oxygen tension distributions in muscle

Characterizing the resistances to O(2) transport from the erythrocyte to the mitochondrion is important to understanding potential transport limitations. A mathematical model is developed to accurately determine the effects of erythrocyte spacing (hematocrit), velocity, and capillary radius on the mass transfer coefficient. Parameters of the hamster cheek pouch retractor muscle are used in the calculations, since significant amounts of experimental physiological data and mathematical modeling are available for this muscle. Capillary hematocrit was found to have a large effect on the PO(2) distribution and the intracapillary mass transfer coefficient per unit capillary area, k(cap), increased by a factor of 3.7 from the lowest (H=0.25) to the highest (H=0.55) capillary hematocrits considered. Erythrocyte velocity had a relatively minor effect, with only a 2.7% increase in the mass transfer coefficient as the velocity was increased from 5 to 25 times the observed velocity in resting muscle. The capillary radius is varied by up to two standard deviations of the experimental measurements, resulting in variations in k(cap) that are <15% at the reference case. The magnitude of these changes increases with hematocrit. An equation to approximate the dependence of the mass transfer coefficient on hematocrit is developed for use in simulations of O(2) transport from a capillary network.     

Growth of Mycobacterium leprae under low oxygen tension

Despite numerous attempts, Mycobacterium leprae has yet to be cultivated in vitro. This organism has been considered as microaerophilic. The effects of various known gas mixtures on the in vitro growth of M. leprae were investigated. A gas mixture containing 2.5% O2 and 10% CO2 was found to be more favourable for the growth of this mycobacterium on artificial medium. Growth was evaluated by three parameters namely cell counts, bacterial ATP and DNA. An optimal growth of M. leprae, as determined by all three parameters, on both liquid and solid media was obtained between 18 and 24 weeks of incubation under optimal gas mixture. Solid medium which contained egg-yolk was relatively more beneficial for in vitro growth than the liquid medium. The cultivated bacilli exhibited some important characteristics specific for M. leprae, including growth in mouse foot-pads. The bacilli gradually lost their power of adaptation to grow on artificial media and did not show any ATP or DNA after about 36 weeks of incubation.