In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique

Mitochondrial oxygen tension (mitoPO₂) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO₂ in vivo exists. Here we report in vivo measurement of mitoPO₂ and the recovery of mitoPO₂ histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO₂ in rat liver in vivo. The results demonstrate mitoPO₂ values of ∼30-40 mmHg. mitoPO₂ was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO₂ distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.     

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.     

Rapid high-throughput assessment of aerobic bacteria in complex samples by fluorescence-based oxygen respirometry

A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given.     

Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling

Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O(2)] has been examined previously, such investigations did not apply rigorous control over [O(2)] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O(2)] as low as 0.1 microM. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O(2)]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.     

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 neutralization 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 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.     

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.     

Comparison of dynamic oxygen electrode methods for the measurement of KLa

The dynamic oxygen electrode method for measuring K_L a requires the use of a dynamic process model. Six models from the literature are described and compared with respect to their accuracy and ease of use. It is shown theoretically that for sufficient accuracy K_L a should be less than the inverse electrode response time. Experimental measurements demonstrate their application to viscous and nonviscous systems. The liquid diffusion film is shown to cause an important measurement lag that can be accounted for by a first-order time delay. Investigation on the influence of the experimental starting conditions show the importance of the gas and hold-up dynamics. A new method is proposed to simplify the K_L a calculation and to eliminate errors caused by starting conditions. This method, which accounts for gas, film, and electrode dynamical effects, requires only a simple semilog plot of the response data.     

Cyclin D1 determines mitochondrial function in vivo

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.     

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.     

In vivo measurement of dynamic rectus femoris function at postures representative of early swing phase

Forward dynamic models suggest that muscle-induced joint motions depend on dynamic coupling between body segments. As a result, biarticular muscles may exhibit non-intuitive behavior in which the induced joint motion is opposite to that assumed based on anatomy. Empirical validation of such predictions is important for models to be relied upon to characterize muscle function. In this study, we measured, in vivo, the hip and knee accelerations induced by electrical stimulation of the rectus femoris (RF) and the vastus medialis (VM) at postures representatives of the toe-off and early swing phases of the gait cycle. Seven healthy young subjects were positioned side-lying with their lower limb supported on air bearings while a 90 ms pulse train stimulated each muscle separately or simultaneously. Lower limb kinematics were measured and compared to predictions from a similarly configured dynamic model of the lower limb. We found that both RF and VM, when stimulated independently, accelerated the hip and knee into extension at these postures, consistent with model predictions. Predicted ratios of hip acceleration to knee acceleration were generally within 1 s.d. of average values. In addition, measured responses to simultaneous RF and VM stimulation were within 13% of predictions based on the assumption that joint accelerations induced by activating two muscles simultaneously can be found by adding the joint accelerations induced by activating the same muscles independently. These results provide empirical evidence of the importance of considering dynamic effects when interpreting the role of muscles in generating movement.     

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.     

Nitrifying activity monitoring and kinetic parameters determination in a biofilm airlift reactor by respirometry

The applicability of batch respirometry, as a simple technique for monitoring off-line nitrifying activity and kinetic parameters, was evaluated using two sets of ammonia and nitrite concentrations. The O₂ uptake rate (OUR) profiles obtained from the assays were adjusted to a substrate inhibition model. The maximum specific ammonia-oxidizing biomass activity (r_Smax) was 0.079 g N-NH₄ ⁺ g VSS^–1 d^–1 with a half saturation coefficient (K_S) of 11 mg N-NH₄ ⁺ l^–1 and an inhibition coefficient (K_i) of 3300 mg N-NH₄ ⁺ l^–1. Besides, the maximum specific value of nitrite-oxidizing activity was 0.082 g N-NO₂ ^– g VSS^–1 d^–1 with a K_S of 4.1 mg N-NO₂ ^– l^–1 and K_i of 1400 mg N-NO₂ ^– l^–1.