Use of spin probe in the determination of oxygen consumption in human neutrophils

A quantitative method of the oxygen consumption rate measurement by human blood neutrophils upon their activation is described. This method is based on the spin exchange determination between 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl spin probe and oxygen molecules from the ESR spectra. Method allows the determination of about 1 micromolar concentrations of oxygen.     

Kinetics of oxygen consumption after a single isometric tetanus of frog sartorius muscle at 20 degrees C

The time-course of the rate of oxygen consumption (QO2) has been measured in the excised frog sartorius muscle after single isometric tetani of 0.1-1.0 s at 20 degrees C. To measure deltaQO2(t), the change in QO2 from its basal level, a novel method was devised, based on the validity in this tissue of the one-dimensional diffusion equation for oxygen, established in the preceding paper. After a tetanus, deltaQO2 reached a peak within 45-90 s, then declined exponentially, and could be well fit by deltaQO2(t) = QO + Q1(epsilon -k1t - epsilon-k2t). tau2 (= 1/k2), which characterized the rise of deltaQO2, was a decreasing function of tetanus duration (range: from 1.1 +/- 0.28 min [nu = 5] for a 0.1-s tetanus, to 0.34 +/- 0.05 min [nu = 8] for a 1.0-sec tetanus). tau1 (= 1/k1), which characterized the decline of deltaQO2, was not dependent on tetanus duration, with mean 3.68 +/- -.24 min (nu = 46). A forthcoming paper in this series shows that these kinetics of deltaQO2 are the responses to impulse-like changes in the rate of ATP hydrolysis. The variation of tau2 with tetanus duration thus indicates the involvement of a nonlinear process in the coupling of O2 consumption to ATP hydrolysis. However, the monoexponential decline of deltaQO2(t), with time constant independent of tetanus duration, suggests that during this phase, the coupling is rate-limited by a single reaction with apparent first order kinetics.     

Luciferase-dependent oxygen consumption by bioluminescent vibrios.

Oxygen uptake due to luciferase in two luminous Vibrio species was estimated in vivo by utilizing inhibitors having specificities for luciferase (decanol) and cytochromes (cyanide). Cyanide titration of respiration revealed a component of oxygen uptake less sensitive to cyanide which was completely inhibitable by low concentrations of decanol. From this it was estimated that in vivo luciferase is responsible for less than 12% (Vibrio harveyi) or 20% (Vibrio fischeri) of the total respiration. From these data in vivo bioluminescent quantum yields are estimated to be not lower than 1.7 and 2.6%, respectively.     

Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.     

Kinetic studies of biodegradation of insoluble compounds by continuous determination of oxygen consumption

Continuous determination of oxygen consumption by electrolytic respirometry has been experimented as a means to study the biodegradation kinetics of scarcely soluble environmental pollutants. The substrates used were the polycyclic aromatic hydrocarbons (PAH), naphthalene, phenanthrene, anthracene, fluoranthene and pyrene. The definition of an appropriate mode of PAH supply, either as crystals or more generally as a solution in a water non-miscible solvent, was found essential for yielding reproducible biodegradation kinetics. In these conditions, for all compounds tested, oxygen determination was found suitable for quantitative evaluation of PAH biodegradation and formation of biomass and soluble metabolites. The study of biodegradation kinetics with this methodology showed that a first phase of exponential growth could be characterized in most cases, followed by a phase of limited growth. Possible mechanisms involved in insoluble substrate uptake are discussed. During exponential growth, the bacteria utilized (although not necessarily exclusively) the PAH solubilized in the aqueous medium.     

Endotoxin-induced liver hypoxia: defective oxygen delivery versus oxygen consumption.

In vivo EPR was used to investigate liver oxygenation in a hemodynamic model of septic shock in mice. Oxygen-sensitive material was introduced either (i) as a slurry of fine particles which localized at the liver sinusoids (pO2 = 44.39 +/- 5.13 mmHg) or (ii) as larger particles implanted directly into liver tissue to measure average pO2 across the lobule (pO2 = 4.56 +/- 1.28 mmHg). Endotoxin caused decreases in pO2 at both sites early (5-15 min) and at late time points (6 h after endotoxin; sinusoid = 11.22 +/- 2.48 mmHg; lobule = 1.16 +/- 0.42 mmHg). The overall pO2 changes observed were similar (74.56% versus 74.72%, respectively). Blood pressures decreased transiently between 5 and 15 min (12.88 +/- 8% decrease) and severely at 6 h (59 +/- 9% decrease) following endotoxin, despite volume replacement with saline. Liver and circulatory nitric oxide was elevated at these times. Liver oxygen extraction decreased from 44% in controls to only 15% following endotoxin, despite severe liver hypoxia. Arterial oxygen saturation, blood flow (hepatic artery), and cardiac output were unaffected. Pretreatment with l-NMMA failed to improve endotoxin-induced oxygen defects at either site, whereas interleukin-13 preserved oxygenation. These site-specific measurements of pO2 provide in vivo evidence that the principal cause of liver hypoxia during hypodynamic sepsis is reduced oxygen supply to the sinusoid and can be alleviated by maintaining sinusoidal perfusion.     

Determination of maximal oxygen consumption in exercising pregnant sheep.

Previous work with pregnant ewes has shown that acute bouts of exercise may cause changes in plasma hormone concentrations, blood flow distribution, and maternal and fetal temperatures. However, most of these studies do not quantify the chosen exercise intensity through measurement of oxygen consumption (VO2). Therefore the purpose of this study was to statistically model the VO2 response of pregnant sheep to treadmill (TM) exercise to determine the exercise intensities (% maximal VO2) of previous studies. Ewes with either single (n = 9) or twin (n = 5) fetuses were studied from 100 to 130 days of gestation. After 1-2 wk of TM habituation, maximal VO2 (VO2max) was determined by measurements of VO2 (open flow-through method) and blood lactate concentration. VO2 was measured as a function of TM incline (0, 3, 5, and 7 degree) and speed (0.8-3.4 m/s). VO2max averaged 57 +/- 7 (SD) ml.min-1.kg-1, and peak lactate concentration during exercise averaged 22 +/- 2 mmol/l. The relationship between VO2 (ml.min-1.kg-1) and incline (INC) and speed (SP) [VO2 = 0.70(INC) + 13.95(SP) + 1.07(INC x SP) - 1.18] was linear (r2 = 0.94). Our findings suggest that most previous research used exercise intensities less than 60% VO2max and indicate the need for further research that examines the effect of exercise during pregnancy at levels greater than 60% VO2max.     

Errors in predicting maximal oxygen consumption in pregnant women.

This study was designed to determine the accuracy of estimated values of maximal heart rate (HRmax) and oxygen consumption (VO2) during pregnancy. We measured HR and maximal VO2 (VO2max) at rest and during cycle (CE) and treadmill exercise (TE) tests with rapidly increasing exercise intensities during gestation and after delivery. Pregnancy was found to affect the linear relationship of HR and %VO2max so that the intercept increases with advancing gestation and the slope decreases. Estimated maximal HR (HRmax, est), 220 - age (yr) x beats/min, overestimated measured HRmax by 8% (CE) and 5% (TE). For VO2max estimated by Astrand's nomogram (VO2max, est1) and by linear extrapolation of submaximal values of HR and VO2 to HRmax, est (VO2max, est2), individual errors were large (SD 17-28%). Mean VO2max, est1 overestimated measured VO2max by 20% during CE but not during TE (-2%) and elicited the erroneous impression that VO2max decreases during CE in pregnancy. Mean VO2max, est2 values were not significantly different from measured VO2max values. This apparent accuracy resulted from two opposing errors: 1) HRmax, est overestimated HRmax, and 2) above 70% VO2max the slope of the HR-%VO2max relationship was significantly reduced. Therefore neither method to estimate VO2max can replace the measurement of VO2max.     

Arachidonate-dependent oxygen consumption in Dictyostelium discoideum

A central feature of the processes of aggregation and differentiation in the cellular slime mould Dictyostelium discoideum is the periodic excitatory cycle. Originally thought to involve primarily fluctuations in cyclic AMP levels, this excitatory cycle has since been shown to involve changes in several other second messengers including cyclic GMP, calcium and inositol trisphosphate. Previous work from this laboratory using specific inhibitors strongly suggested a role for eicosanoids in this stimulus-response process. Production of eicosanoids from fatty acid precursors is an oxygen-consuming process. In this paper, we report on oxygen consumption measurements in intact D. discoideum cells and in cell extracts. We demonstrate the existence of an azide-insensitive component of oxygen consumption which can be stimulated by the addition of arachidonate and other polyunsaturated fatty acids, and at least partially inhibited by meclofenamate and eicosatetraynoic acid, both of which block eicosanoid biosynthesis in higher organisms. These observations provide further evidence for the existence of an eicosanoid-metabolizing system in D. discoideum.     

Below-halocline oxygen consumption in the Kattegat

Sediment and seston oxygen consumption rates below the sharp halocline in the south-eastern part of the shallow Kattegat were measured and compared to calculated rates of carbon addition through the halocline. The mean rate of decrease in deep-water oxygen concentrations between March and September 1988 was 1.0 ml O₂ M^–3 h^–1. Measurements of benthic oxygen uptake using laboratory-incubated sediment cores from depths 30 m gave a mean value of 7.8 ml O₂ m^–2 h^–1. Below-halocline water (from 20 m, 30 m and 1 m above bottom) incubated in bottles showed oxygen consumption rates varying from 0.5 ml O₂ m ^–3 h^–1 in March to 2.8 ml O₂ M^–3 h^-1 in late August. The sum of benthic and deep-water oxygen consumption was equivalent to a mean oxygen decrease rate of 1.7 ml O₂ m^–3 h^–1 below the halocline. Of the total oxygen consumption below the halocline 65% was due to oxygen up-take in the water and 35% was due to benthic oxygen consumption. The sum of oxygen consumption measured in sediment cores and in bottles corresponds to a carbon utilisation of 80.1 g C m^–2 (respiratory quotient (RQ), assumed 1.0 and 1.4 for water and sediment, respectively), while the decrease in deep-water oxygen concentration was equivalent to 43.0 g C m^–2 (RQ assumed = 1.0). Using published values for the external N loading (including deep-water supply), ¹⁵NO₃-uptake, ¹⁴CO₂-uptake in combination with % ¹⁵NO₃-uptake of total ¹⁵N-uptake (nitrate, ammonia and urea) and a Redfield C/N ratio of 6.6, rates of carbon addition (new or export production) through the halocline were calculated to 31.9, 46.7 and 36.3 g C m^–2, respectively, with a mean value of 38.3 g C m^–2 for the 8 month period March–September. This is somewhat less than the value (50.5 g C m^–2) calculated from a published empirical relationship between total and export production. The fact that the calculated carbon addition through the halocline was appreciably less than the carbon equivalent of the measured below-halocline respiration may be an effect of sediment focusing (horizontal transport of sedimenting material to deeper areas), since the bottom area below the halocline is much smaller than the total area of the Kattegat. A lower observed decrease in the oxygen concentration below the halocline compared to the sum of measured sediment and deep-water oxygen consumption on the other hand indicates oxygen supply to below-halocline waters through advection and/or vertical entrainment.