Oxidative stress and methods used for hydroxyl radical determination

Understanding the role of oxidative stress in brain as well as developing medical strategies to reduce its damaging potential in the aging process and pathogenesis of cancer, neurological diseases like Alzheimer’s diseases and Parkinson’s diseases and other incurable illnesses is an important direction in medicine and biochemistry over the world. This review outlines the processes by which hROS may be formed, their damaging potential and determinations methods. Also, the questions upon the nature of reactive hROS in a Fenton (like) system plays a crucial role will be addressed on this part and several lines of evidences will be presented in order to clarify this issue. Highly reactive hydroxyl radicals (hROS) have been implicated in the etiology of many diseases, therefore monitoring of hROS should be extremely helpful to further investigate and understand the role of hROS in the pathogenesis of neurological disorders and to develop medical strategies to reduce the damaging potential of hROS. The very short half-life of OH• requires the use of trapping agents such as salicylic acid or phenylalanine for detection, but their hydroxylated derivatives are either unstable, or implicated as reactant in biochemical processes. Based on already successfully in vitro and in vivo work done in our group in the past two decades, we decided to use sodium terephthalic acid as a trapping agent, the hydroxylation of which yields only one stable and highly fluorescent isomer, 2-hydroxyterephthalate (OH-TA)

Study of OH• Radicals in Human Serum Blood of Healthy Individuals and Those with Pathological Schizophrenia

The human body is constantly under attack from free radicals that occur as part of normal cell metabolism, and by exposure to environmental factors such as UV light, cigarette smoke, environmental pollutants and gamma radiation. The resulting “Reactive Oxygen Species” (ROS) circulate freely in the body with access to all organs and tissues, which can have serious repercussions throughout the body. The body possesses a number of mechanisms both to control the production of ROS and to cope with free radicals in order to limit or repair damage to tissues. Overproduction of ROS or insufficient defense mechanisms leads to a dangerous disbalance in the organism. Thereby several pathomechanisms implicated in over 100 human diseases, e.g., cardiovascular disease, cancer, diabetes mellitus, physiological disease, aging, etc., can be induced. Thus, a detailed investigation on the quantity of oxygen radicals, such as hydroxyl radicals (OH•) in human serum blood, and its possible correlation with antioxidant therapy effects, is highly topical. The subject of this study was the influence of schizophrenia on the amount of OH• in human serum blood. The radicals were detected by fluorimetry, using terephthalic acid as a chemical trap. For all experiments the serum blood of healthy people was used as a control group

Evaluating the use of 3'-(p-Aminophenyl) fluorescein for determining the formation of highly reactive oxygen species in particle suspensions

<p>Abstract</p> <p>Background</p> <p>Given the importance of highly reactive oxygen species (hROS) as reactants in a wide range of biological, photochemical, and environmental systems there is an interest in detection and quantification of these species. The extreme reactivity of the hROS, which includes hydroxyl radicals, presents an analytical challenge. 3'-(<it>p</it>-Aminophenyl) fluorescein (APF) is a relatively new probe used for measuring hROS. Here, we further evaluate the use of APF as a method for the detection of hydroxyl radicals in particle suspensions.</p> <p>Results</p> <p>Particle-generated hROS can be quantified with an estimated detection limit of 50 nM. Measurements of hROS in two National Institute of Standards and Technology (NIST 2709 and 2710) soil suspensions and a pyrite suspension show non-linear particle dose-response curves for hROS generation. APF can also be used in solutions containing no dissolved molecular oxygen (O₂) to determine the role of O₂in the formation of hROS. Results confirm that O₂is mechanistically important in the formation of hROS by dissolved ferrous iron and in pyrite suspensions.</p> <p>Conclusion</p> <p>Given the non-linear dose-response curves for particle generation of hROS, we recommend using several particle loadings in experiments aimed to compare particles for their hROS generation potential. The method presented here is specific to hROS and simple to perform. The analysis can be conducted in mobile labs as only basic laboratory equipment is required.</p

Calprotectin (S100A8/S100A9) and Myeloperoxidase: Co-Regulators of Formation of Reactive Oxygen Species

Inflammatory mediators trigger polymorphonuclear neutrophils (PMN) to produce reactive oxygen species (ROS: O2-, H2O2, ∙OH). Mediated by myeloperoxidase in PMN, HOCl is formed, detectable in a chemiluminescence (CL) assay. We have shown that the abundant cytosolic PMN protein calprotectin (S100A8/A9) similarly elicits CL in response to H2O2 in a cell-free system. Myeloperoxidase and calprotectin worked synergistically. Calprotectin-induced CL increased, whereas myeloperoxidase-triggered CL decreased with pH > 7.5. Myeloperoxidase needed NaCl for CL, calprotectin did not. 4-hydroxybenzoic acid, binding ∙OH, almost abrogated calprotectin CL, but moderately increased myeloperoxidase activity. The combination of native calprotectin, or recombinant S100A8/A9 proteins, with NaOCl markedly enhanced CL. NaOCl may be the synergistic link between myeloperoxidase and calprotectin. Surprisingly- and unexplained- at higher concentration of S100A9 the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function. We propose that the ∙OH is predominant in ROS production by calprotectin, a function not described before

New chemical and biological insights into the chemistry of highly reactive oxygen species

Zsfassung in dt. SpracheDer Schwerpunkt der vorliegenden Arbeit ist einerseits die Entwicklung einer neuen zuverlässigen Methode, um hoch reaktive Sauerstoffverbindungen direkt im Gehirn mittels Mikrodialysesonden messen zu können, und andererseits die chemisch-mechanistische Untersuchung der Fenton Reaktion unter physiologische Bedingungen, da diese höchstwahrscheinlich den Grund für die Bildung dieser Verbindungen im Gehirn darstellt. Des Weiteren werden die Entwicklung und in vivo Anwendung von zwei weiteren Methoden beschrieben, wobei eine zur Bestimmung von reaktivem Eisen (II) sowie freigesetztem Eisen (III) und die andere zur Messung von Wasserstoffperoxid herangezogen werden kann. Hochreaktive Sauerstoffverbindungen (hROS), die im Allgemeinen entweder als Hydroxylradikale oder Eisen(IV) Verbindungen auftreten, wird eine entscheidende Rolle bei der Entstehung von neurodegenerativen Krankheiten und beim Alterungsprozess zugeschrieben. Aufgrund ihrer sehr hohen Reaktivität ist eine direkte Messung derzeit unmöglich, und es muss auf indirekte Methoden, die meist auf der Hydroxylierung von Aromtaten basieren, zurückgegriffen werden. Die am häufigsten benutzte Methode zu ihrer Messung greift auf die Hydroxylierung von Salicylsäure zurück. Da diese jedoch einerseits biologisch aktiv ist, und andererseits ihre, je nach chemischer Umgebung unterschiedlichen, Hydroxylierungsprodukte keine einwandfreie Quantifizierung erlauben, wurden immer wieder andere Substanzen als Alternative vorgeschlagen. In dieser Arbeit wurde die Hydroxylierung von Terephthalsäure (TA), deren Produkt die äußerst gut fluoreszierende 2-Hydroxy-Terephthalsäure (OH-TA) ist, sowohl nach chemischen als auch nach biologischen Kriterien untersucht. Es konnte festgestellt werden, dass TA weder die Neurotransmitter Glutamat, Aspartat, [gamma]-Aminobuttersäure und Taurin im Striatum der Ratte beeinflusst, noch chemische Artefakte einer genauen Quantifizierung von hROS im Wege stehen. Die in vivo Stimulierung des Striatums mit drei unterschiedlichen Konzentrationen des Neurotoxins Kainsäure ergab eine Dosis abhängige Bildung von hROS, womit das Funktionieren der Methode bestätigt werden konnte. Auf der anderen Seite konnte gezeigt werden, dass neuroprotektive Substanzen die hROS Bildung unterdrückten. Von praktischem Vorteil ist weiters, dass OH-TA gemeinsam mit o-Phthaldialdehyd derivatisierten Aminosäuren mittels HPLC gemessen werden kann, und so keine zusätzlichen Probemengen gebraucht werden. Wird die hROS Bildung durch die Fenton Reaktion in einer der extrazellulären Flüssigkeit nachempfundenen Salzlösung beobachtet, so ist die Menge vom gebildeten OH-TA unabhängig von der TA Konzentration.Diese Beobachtung kann nicht mit der Bildung von freien Hydroxylradikalen in Übereinstimmung gebracht werden, sondern ausschließlich mit einem "krypto" Radikal oder einer Eisen(IV) Verbindung erklärt werden. Diese Annahme konnte mit Experimenten in Kaliumacetetat Puffer mit Hilfe eines durch kinetische Messungen unterstützen Reaktionsschemas bestätigt werden. Um die Fenton Reaktion auch in vivo messen zu können war es notwendig mit kleinsten Probemengen eine Eisen -und Wasserstoffperoxidbestimmung durchführen zu können. Die bereits bekannte, aber für Mikrodialyseexperimente nicht brauchbare, photometrischen Eisenbestimmung mit Hilfe von Bathophenanthrolin wurde modifiziert, um Eisenmengen im nano-molaren Bereich unter Verwendung der nicht selektiven UV-Bande bei 385 nm mittels HPLC messen zu können. Zusätzlich wurde Wasserstoffperoxid mit Hilfe von TA und Fe(II)EDTA bestimmt. Es konnte gezeigt werden, dass die in vivo Stimulierung mit 6-Hydroxy-dopamin, das als einfaches Tiermodell für die Parkinson`sche Krankeit gilt, zu einer massiven Eisen- und Wasserstoffperoxidfreisetzung führt, wobei für die hROS Bildung ausschließlich die Menge an aktivem Eisen (II) entscheidend ist.The main emphasis of this work was to develop and test a new reliable method to detect in vivo highly reactive oxygen species (hROS) directly in the rat's striatum using microdialysis and to analyse a Fenton system in a chemical environment approximated to the extra cellular fluid utilising kinetic measurements. Beside that, two new methods to detect iron and H2O2 in the nano-molar range with sample volumes of few micro-litres via HPLC are introduced. Finally the results of in vivo experiments using a simple model of Parkinson's disease, combing all methods developed in this work are presented.Sodium terephthalate was validated as a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), lacking the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA2-) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions, accompanied by chemical in vitro investigations.Derivatisation with o-phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram, with a detection limit of 0.5 femtomol/µl of OH-TA in microdialysis samples. In microdialysis experiments the neurotoxin kainate was shown to evoke hROS formation in a dose-dependent manner.The presence of TA2- in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death 'ex vivo' showed TA2- to be non-toxic at concentrations up to 1.0*10-3 mol/L . The in vitro results based on kinetical measurements indicate a mechanism in the Fenton system (Fe2+ + H2O2) without the involvement of a free hydroxyl radicals, whereby TA2- forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer. To measure all relevant compounds involved in the Fenton reaction also in vivo, two new methods, based on known procedures, to measure iron and H2O2 were developed. Iron was detected by HPLC using the non selective UV band at 285 nm of the Fe(II)-bathophenanthroline complex, whereas H2O2 was measured by utilising Fe(II)EDTA and TA. In the end of this work the in vivo results regarding hROS, active and released iron and H2O2 using the stimulation with 6-hydroxy-dopamine (6-OHDA) which can be considered a simple model of PD, are presented. It is shown that the massive formation of hROS after stimulation is mainly caused by active Fe(II) and not by total iron or H2O2.11

Development of an Online Fluorescence Method for near real time in vivo monitoring of Hydroxyl Radicals in rats

Hydroxyl radicals have been implicated in the etiology of many diseases, therefore on line monitoring of hROS should be extremely helpful to further investigate and understand the role of hROS in the pathogenesis of neurological disorders and to develop medical strategies to reduce the damaging potential of hROS. Furthermore, while the use of the HPLC is limited in terms of time resolution (sampling time could not be reduced below 10 min) the on line system allows real-time measurements, which is crucial for understanding the chemical events involved in physiological and pathological processes. Therefore, the main emphasis of this work was to investigate hROS in vivo on line by using a simple and well characterized animal model of excitotoxic damage based on the application of a high concentration (1 mM and 500μM) of the non-NMDA glutamate receptor agonist, kainate (KA), to the neostriatum in freely moving animals through the dialysis probe. For this purpose a highly sensitive fluorescence detector equipped with a capillary flow cell, coupled directly to the rat striatal microdialysis system, was successfully developed and employed for continuous on line determination of hROS under in vivo conditions. Comparing with the HPLC or other analytical methods which are used for hROS detection, the presented method has provided significant advantages in terms of its sensitivity and simplicity. Further, due to its better temporal resolution and high precision, this method could find a wide application in understanding of hROS chemical events involved in some physiological and pathological processes and might also lead to a human application