Measurement of Mitochondrial Respiration in Platelets.

Platelet mitochondria can be used in the study of mitochondrial dysfunction in various complex diseases and can help in finding biological markers for diagnosing the disease, monitoring its course and the effects of treatment. The aim of this chapter was to describe in detail the method of measuring mitochondrial respiration in platelets using high-resolution respirometry. The described method was successfully used for the study of mitochondrial dysfunction in neuropsychiatric diseases.

A Rapid Mercury Droplet Electrode Polarography Method for Determination of Blood Lead Level in Lead Poisoned People.

BACKGROUND: Lead is a hazardous heavy metal, which causes many problems in the human body. Unfortunately, recent reports showed that smugglers and opium sellers add lead to drugs during the production procedure in order to increase its weight and cost. PURPOSE: The aim of this study was development of a rapid and accurate method for measurement of blood lead levels (BLL) in the oral and inhaled opiate abuser people. METHODS: BLL in samples obtained from the oral and inhaled opium addicted patients referring to Sina Hospital in Tabriz, Iran, during 2017 was compared with healthy control group (N=15). The wet digestion method was used to prepare whole blood and Mercury Droplet Electrode Polarography (MDEP) method was utilized for measurement of the lead content of digested samples. RESULTS: Results showed that there were significant differences between the BLL of samples obtained from oral (17.12±74.61 µg/dL, p<0.0003) and inhaled (19.33±2.257 µg/dL, p<0.0001) opium addicted groups in comparison with healthy control group (4.669±0.3367 µg/dL). CONCLUSION: Based on the results of this study it was observed that BLL in opium addicted people needs to be measured as soon as possible. Furthermore, screening of blood lead concentrations in opium-addicted people with a rapid and accurate MDEP method is very necessary and important.

Implementation of a logarithmic division-of-focal-plane polarimeter to quantify changes in collagen alignment at varying levels of illumination.

We examine the impact of illumination, aperture, and sample thickness on two division-of-focal-plane (DoFP) polarimeters, one created using a standard 3 T pixel and the other with a forward-biased, logarithmic pixel. Across all measured metrics the logarithmic DoFP polarimeter was better able to track real-time changes in collagen alignment than the standard DoFP polarimeter.

Differential pulse polarographic investigation of micafungin and anidulafungin using a dropping mercury electrode.

The electrochemical behavior of the echinocandin antifungals anidulafungin (AF) and micafungin (MF) has been investigated by differential pulse polarography (DPP). The measurements were carried out in a supporting electrolyte solution consisting of Britton-Robinson buffer and methanol at various substance concentrations and pH values. An amperometric cell with a three electrode system consisting of a dropping mercury electrode (DME) as working electrode, an auxiliary platinum electrode and an Ag/AgCl reference electrode was used in all experiments. AF was electrochemically reduced at potentials between -1.3 and -1.5 V. MF showed a first reduction peak (a) between -1.0 and -1.4 V and a second peak (b) between -1.5 and -1.8 V. A strong pH-dependence was observed, with optimal results at pH 2.0-3.0 for the AF peak, pH 2.0 for the MF peak (a) and pH 5.0 for the MF peak (b). A linear correlation between the concentration and the peak current has been demonstrated for all reduction peaks. MF peak (a) showed a similar behavior to the AF peak regarding shape, peak current and pH-dependence. Therefore, it can be assumed that both reductions are based on the same mechanism, a two-step reduction of the N-acyl group.

Voltammetric analysis of dantrolene and its active metabolite with indomethacin in rat plasma.

AIM: Differential pulse polarography was used for the concurrent analysis of the coadministered dantrolene (DAN) and indomethacin (IND) in plasma. MATERIALS & METHODS: DAN and IND, Hanging mercury drop electrode and Britton-Robinson buffer at pH 5 were used. In plasma, cathodic reduction of DAN nitro group and its active metabolite at -0.2 V was done. IND was analyzed after carbonyl group reduction at -1.1 V. RESULTS: Drugs determination in rat plasma with good recoveries and low limit of quantitation was done. Application to trace analysis of drugs in rat plasma was done with Cmax and Tmax determination. CONCLUSION: This technique shows high sensitivity, simplicity and low cost. The method is US FDA validated and it is applicable to human level.

Evaluation of Respiration with Clark-Type Electrode in Isolated Mitochondria and Permeabilized Animal Cells.

In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the classic end points that can be assessed is oxygen consumption, which can be performed under controlled yet artificial conditions. Oxygen is the terminal acceptor in the mitochondrial respiratory chain, namely, at an enzyme called cytochrome oxidase, which produces water in the process and pumps protons from the matrix to the intermembrane space. Several techniques are available to measure oxygen consumption, including polarography with oxygen electrodes or fluorescent/luminescent probes. The present chapter will deal with the determination of mitochondrial oxygen consumption by means of the Clark-type electrode, which has been widely used in the literature and still remains to be a reliable technique. We focus our technical description in the measurement of oxygen consumption by isolated mitochondrial fractions and by permeabilized cells.

High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria.

Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O2 flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O2 concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O2 limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O2 flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.

High-Throughput Analysis of Mitochondrial Oxygen Consumption.

Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe , standard microtiter plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.

Assessing Mitochondrial Bioenergetics by Respirometry in Cells or Isolated Organelles.

Mitochondrial oxidative phosphorylation is central for generating ATP and maintaining energy homeostasis in most eukaryotic cells. The ex vivo measurement of mitochondrial oxygen consumption rates in intact cells or isolated organelles is a valuable approach to assess mitochondrial bioenergetics in various experimental conditions. In this chapter, we describe several step-by-step protocols for measuring mitochondrial respiration in intact cells, permeabilized cells (in situ mitochondria), and isolated organelles using both Clark-type polarographic oxygen electrode devices and the newly developed oxygen-sensing fluorophore-based Seahorse technology.

Predictive value of hypoxia in advanced head and neck cancer after treatment with hyperfractionated radio-chemotherapy and hypoxia modification

Purpose. Hypoxia has predictive value in head and neck cancer (HNC). It has been well described, albeit in a small number of clinical Centres. The aim of this study was to describe our experience using the polarographic probe technique to assess the predictive value of tumour oxygenation in patients with advanced HNC treated with hyperfractionated radio-chemotherapy. Hypoxia modification was induced using percutaneous spinal cord stimulation (SCS). Methods/patients. Male patients (n = 12; stage IVb n = 8; IVa n = 4; mean age 58: range 46-70 years) with advanced HNC were evaluated. Planned therapy was hyperfractionated-radiotherapy, oral tegafur (precursor of 5-fluorouracil) and hypoxia modification using SCS. Pre-treatment analyses included: haemoglobin levels and tumour oxygenation (using the Eppendorf polarographic probe device). Oxygenation was expressed as median-pO2 (in mmHg) and hypoxia as the percentage of pO2 values ≤5 mmHg (HP5) and ≤2.5 mmHg (HP2.5). Results. Lower haemoglobin levels were directly correlated with median pO2 (p = 0.017) and inversely correlated with HP5 (p = 0.020) and more advanced stages (IVb vs. IVa; p = 0.028). Patients who subsequently developed systemic metastasis had tumours that were more hypoxic, with lower median pO2 (p = 0.036) and higher HP5 (p = 0.036). The subgroup of patients with HP2.5 above the median (the most hypoxic tumours) had lower loco-regional control (p = 0.027), cause-specific survival (p = 0.008), and overall survival (p = 0.008). Conclusions. Higher tumour hypoxia showed predictive value in HNC in our study, and was significantly associated with lower overall survival, cause-specific survival, and loco-regional control. Tumour hypoxia determination could be used to select patients who would most benefit by hypoxia modification during chemo-radiotherapy of HNC (AU)

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Oxygen-Sensing Methods in Biomedicine from the Macroscale to the Microscale.

Oxygen monitoring has been a topic of exhaustive study given its central role in the biochemistry of life. The ability to quantify the physiological distribution and real-time dynamics of oxygen from sub-cellular to macroscopic levels is required to fully understand the mechanisms associated with both normal physiology and disease states. This Review will present the most significant recent advances in the development of oxygen-sensing materials and techniques, including polarographic, nuclear medicine, magnetic resonance, and optical approaches, that can be applied specifically for the real-time monitoring of oxygen dynamics in cellular and tissue environments. As some of the most exciting recent advances in synthetic methods and biomedical applications have been in the field of optical oxygen sensors, a major focus will be on the development of these toolkits.

Toxicity assessment of 2,4-D and MCPA herbicides in primary culture of fish hepatic cells.

In this study, we used primary cultures of fish hepatic cells as a tool for evaluating the effects of environmental contamination. Primary hepatic cell cultures derived from the subtropical fish Metynnis roosevelti were exposed to different concentrations (0.275, 2.75 and 27.5 µg L(-1)) of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA). Cellular respiratory activity was evaluated by polarography using three substrates: 0.5 M glucose, 0.5 M succinate and 0.5 M α-ketoglutarate. Significant changes were observed in cellular oxygen consumption with 0.5 M α-ketoglutarate. Even at low concentrations, 2,4-D and MCPA were potent uncouplers of oxidative phosphorylation. Primary cultures of M. roosevelti liver cells may provide a useful tool for the evaluation of environmental contaminant effects. A review of regulations regarding permitted concentrations of these herbicides is needed.

Oxygen permeability measurements of contact lenses: a proposal for accuracy improvement.

Contact lens are a widespread medical device. In view of the importance of a proper oxygenation of the cornea, new materials are continuously being tested, with a high permeability to oxygen. Taking into account the limitations of the methods for testing soft contact lenses, as presented in the relevant international standards, this paper focuses on the polarographic method and on the approach of measuring oxygen permeability of stacked contact lenses. The effect of the interspersed saline solution layers on the measurable permeability of the stack is considered, using Fick's law of diffusive flux, and a proposal for accuracy improvement in oxygen permeability measurements is presented.

Monitoring in microvascular tissue transfer by measurement of oxygen partial pressure: four years experience with 125 microsurgical transplants.

In a prospective study, the characteristics and benefit of an invasive measurement of oxygen partial pressure (pO(2)) with the aid of a polarographic sensor were investigated in 125 microsurgical reconstructions of the head and neck area over a period of 45 months. Measurements were performed over 96 h in eight different types of microsurgically revascularized flaps for extra- and intraoral reconstructions and were evaluated separately for each flap type. Of 125 reconstructions the system indicated malperfusion in 18 cases. Salvage surgery was performed in 17 cases due to venous thrombosis (6 cases), arterial thrombosis (3 cases), a combination of arterial and venous thrombosis (2 cases), rheological problems (3 cases), venous insufficiency by hematoma (2 cases) and kinking of vessels (1 case). In 10 cases salvage surgery was successful, 7 flaps were lost despite salvage surgery. In all these cases, the polarographic probe indicated the necessity of salvage surgery correctly. After 96 h no malperfusion was seen. Postoperatively, a common and characteristic development of the oxygen partial pressure in different types of flaps was seen. Initially, a clear increase of pO(2) could be measured. During 96 h, a slow decrease of pO(2) was observed. In conclusion polarographic measurement of pO(2) can be an excellent apparative supplement for the postoperative clinical control of microsurgically revascularized transplants. In buried flaps, this technique represents the only reliable method for transplant monitoring.

In vivo oxygen uptake into the human cornea.

PURPOSE: We provide a new procedure to quantify in situ corneal oxygen uptake using the micropolarographic Clark electrode. METHODS: Traditionally, upon placing a membrane-covered Clark microelectrode onto a human cornea, the resulting polarographic signal is interpreted as the oxygen partial pressure at the anterior corneal surface. However, the Clark electrode operates at a limiting current. Hence, oxygen flux is directly detected rather than partial pressure. We corrected this misunderstanding and devised a new analysis to quantify oxygen uptake into the cornea. The proposed analysis is applied to new polarographic data for 10 human subjects during open-eye oxygen uptake. RESULTS: Average open-eye corneal oxygen uptake over 10 subjects is approximately 11 µL/(cm(2) h), approximately five times larger than the average reported by researchers who invoke the original mathematical analysis. Application of the classical interpretation scheme to our experimental data also garners uptake values that are approximately a factor of three to five times smaller than those obtained with our new procedure. CONCLUSIONS: The classical procedure originally developed by Fatt and colleagues misinterprets the behavior of the Clark microelectrode. We corrected the analysis of the in situ polarographic technique to provide a simple yet rigorous procedure for analyzing both previous data in the literature and those newly obtained. Our proposed interpretation scheme thus provides a reliable tool for in vivo assessment of corneal oxygen uptake.

Comparison of two probe designs for determining intraocular oxygen distribution.

INTRODUCTION: Alterations in intraocular oxygen levels are important contributors to, or indications of, ocular disease. Polarographic electrodes and fibre-optic sensors (optodes) have been used to measure oxygen and to map the distribution of oxygen in animal models and in human eyes. A recent study reported the use of a commercial electrode to compare oxygen distribution in the vitreous of patients undergoing vitrectomy related to central retinal vein occlusion, macular hole or preretinal membrane. The results of this study were at variance with previous measures of oxygen distribution in the human vitreous using polarographic or optical sensors. To resolve this discrepancy, the present study compared measurements made in vitro or in animal eyes, using the electrode employed in the previous study or a fibre-optic sensor of a different design. STUDY DESIGN: Comparative in vitro and in vivo measurements. RESULTS: In vitro, the two devices reported similar levels of oxygen, although the electrode consistently detected levels above the calculated values. In rabbit eyes, the electrode had a slow response time and was unable to detect oxygen gradients that were readily measured by the smaller optode. When the electrode was inserted into an eye of similar size to the human eye, the reference thermistor measured the temperature outside the eye, not in the vitreous. CONCLUSIONS: The design of the electrode used in the previous study makes it unsuitable for measurements of oxygen distribution in the eye.

Analysis of throughput for multilayer infrared meanderline waveplates.

A meanderline wave retarder is a unique type of frequency-selective-surface (FSS) that enables a change in the state of optical polarization. The principles of operation are very similar to a typical crystalline waveplate, such that the artificially structured meanderline array has both 'slow' and 'fast' axes that provide a phase offset between two orthogonal wave components. In this paper, we study the behavior and response of multilayered meanderline quarter-wave retarders designed for operation at 10.6 mum wavelength (28.28 THz). It will be shown that meanderline quarter-wave plates with more than a single layer exhibit improved transmission throughput at infrared frequencies due to impedance matching, similar to a multilayer optical film coating. Numerical data, both from simulations and measurements, are presented to validate this claim.

Hyperbaric hyperoxia and normobaric reoxygenation increase excitability and activate oxygen-induced potentiation in CA1 hippocampal neurons.

Breathing hyperbaric oxygen (HBO) is common practice in hyperbaric and diving medicine. The benefits of breathing HBO, however, are limited by the risk of central nervous system O2 toxicity, which presents as seizures. We tested the hypothesis that excitability increases in CA1 neurons of the rat hippocampal slice (400 microm) over a continuum of hyperoxia that spans normobaric and hyperbaric pressures. Amplitude changes of the orthodromic population spike were used to assess neuronal O2 sensitivity before, during, and following exposure to 0, 0.6, 0.95 (control), 2.84, and 4.54 atmospheres absolute (ATA) O2. Polarographic O2 electrodes were used to measure tissue slice PO2 (PtO2). In 0.95 ATA O2, core PtO2 at 200 microm deep was 115±16 Torr (mean±SE). Increasing O2 to 2.84 and 4.54 ATA increased core PtO2 to 1,222±77 and 2,037±157 Torr, respectively. HBO increased the orthodromic population spike amplitude and usually induced hyperexcitability (i.e., secondary population spikes) and, in addition, a long-lasting potentiation of the orthodromic population spike that we have termed "oxygen-induced potentiation" (OxIP). Exposure to 0.60 ATA O2 and hypoxia (0.00 ATA) decreased core PtO2 to 84±6 and 20±4 Torr, respectively, and abolished the orthodromic response. Reoxygenation from 0.0 or 0.6 ATA O2, however, usually produced a response similar to that of HBO: hyperexcitability and activation of OxIP. We conclude that CA1 neurons exhibit increased excitability and neural plasticity over a broad range of PtO2, which can be activated by a single, hyperoxic stimulus. We postulate that transient acute hyperoxia stimulus, whether caused by breathing HBO or reoxygenation following hypoxia (e.g., disordered breathing), is a powerful stimulant for orthodromic activity and neural plasticity in the CA1 hippocampus.

Brain tissue oxygen pressure monitoring in awake patients during functional neurosurgery: the assessment of normal values.

Local brain tissue oxygen (ptiO2) monitoring is frequently applied in patients at risk for cerebral ischemia. To identify ischemic thresholds, the normal range of local brain tissue oxygen pressure (ptiO2) values needs to be established. Ideally, such normal values are determined in healthy and awake subjects, so as to eliminate the possible influences of anesthetics on cerebral physiology or ptiO2. Thus far, however, such measurements have not been conducted, and to fill this void, we determined the ptiO2 values in normal white matter of awake patients undergoing functional stereotactic brain surgery. In 25 otherwise healthy patients, who underwent functional neurosurgery for treatment of a refractory movement disorder under local anesthesia, the ptiO2 of white matter was recorded continuously using a polarographic Clark type electrode monitoring system. Preoperative screening ruled out cognitive dysfunction or structural cerebral lesions. Reliable intraoperative ptiO2 values were obtained in 22 patients. After an adaptation period of 118+/-35 min (range, 47-171 min), we found an average normal ptiO2 of 22.6+/-7.2 mm Hg in the frontal white matter. In 11 patients, ptiO2 measurements were continued postoperatively for 24 h. During this period, a similar normal ptiO2 value of 23.1+/-6.6 mm Hg was found. No iatrogenic complications occurred. In conclusion, the normal ptiO2 of cerebral white matter is most likely lower than previously assumed. Further, the long adaptation time renders this widely applied monitoring instrument unreliable in detecting ischemia early after insertion and limits its usefulness for intraoperative monitoring.

Chemometric determination of rabeprazole sodium in presence of its acid induced degradation products using spectrophotometry, polarography and anodic voltammetry at a glassy carbon electrode.

Chemometric stability indicating methods are presented for the determination of rabeprazole sodium in presence of its acid induced degradation products using spectrophotometry, differential pulse polarography and differential pulse anodic voltammetry at a glassy carbon electrode. The applied chemometric techniques are multivariate ones including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS). A difference spectrophotometric (DeltaA) method has also been applied. To develop the multivariate calibrations, a training set was used, consisting of 20 mixture solutions of rabeprazole sodium and its degradation products. These mixtures show percentage degradation ranging from 0.5-65%, 0.5-95% and 0.6-75% for the spectrophotometric, polarographic and anodic voltammetric calibrations, respectively. The UV absorbances were recorded in 0.1 M NaOH within the wavelength range 220-340 nm at 2 nm intervals. The polarograms and anodic voltammograms were recorded in Britton-Robinson buffer (pH 8.0) within the potential range -500 to -1508 and 400 to 1192 mV at 6 mV intervals with a pulse amplitude of -100 and 50 mV, sweep rate of 15 and 10 mV s(-1) and pulse interval of 0.4 and 0.6 s for the polarographic and anodic voltammetric methods, respectively. All the studied methods have been validated and successfully applied to the determination of rabeprazole sodium in tablet dosage form. The results were statistically compared to those obtained using a published HPLC method. No significant difference has been found.