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

Development of an online fluorescence method for near real time in vivo monitoring of hydroxyl radicals in rats

Trotz intensiver Bemühungen existiert bis heute keine on line Methode, um Reaktionen hochreaktiven Sauerstoffverbindungen in einer Echtzeitmessung und wenn möglich in vivo zu untersuchen. Das wichtigste Ziel meiner Arbeit war es daher die Entwickelung einer on line Methode zu entwickeln, mit welcher hoch reaktive Sauerstoffverbindungen in Gehirn von Versuchstieren mittels Mikrodialysesonde gemessen werden können. Viele neurotoxische Prozesse hängen in hohem Maße mit dem Auftreten hochreaktiver Sauerstoffverbindungen (hROS), die als freie oder gebundene Hydroxylradikale oder als Fe(IV)- Sauerstoff Verbindungen vorliegen, zusammen. Hydroxyl Radikale spielen bei vielen neurodegenerativen Krankheiten eine entscheidende Rolle, in generell sie spielen eine entscheidende Rolle im Alterungsprozess, auch während eines Herzstillstandes treten derartige Verbindungen auf. Die Fenton Reaktion gilt als wahrscheinlichste Ursache der Entstehung von hROS. Ein Hauptproblem bei der Aufklärung der biochemischen Mechanismen, die zur Radikal- Bildung führen, ist die kurze (ca. 10-9 s) Lebensdauer der Radikale. Da diese eine direkte Messung unmöglich machen, musste auf geeignete chemische Fallen zurückgegriffen werden, die durch die Hydroxyl -Radikale hydroxyliert werden. Terephthalsäure wurde von Freibichler et al. als alternative Methode vorgeschlagen, da sie aufgrund ihrer Geometrie nur ein definiertes Hydroxy-Derivat zulässt, welches ausreichend stabil ist und ausgezeichnete Fluoreszenzeigenschaften besitzt. Diese neuartige Methode für on line Bestimmung in vivo von hochreaktiven Sauerstoffverbindungen wurde in Zusammenarbeit mit Prof. Laura Della Corte an der medizinischen Universität Florenz mittels eines Fluorescence Detektor entwickelt. Die on line Stimulierung des Stratiums wurde mit zwei unterschiedlichen Konzentrationen der Kainsäure durchgeführt. Weitere Untersuchunge erfolgten mit Hilfe von HPLC an hochreaktiven Sauerstoffverbindungen der Dialysate. In letzten Teil der vorliegenden Arbeit wird eine photometrische Methode für Eisenbestimmung in vitro mit Hilfe von Bathophenanthroline detailiert beschrieben. Die entsprechenden Bestimmungen von Eisenmengen im nano-molaren Bereich wurden unter Verwendung der selektiven Bande 535 nM mittels U-VIS Detector durchgeführt. Eine weiterführende Anwendung dieses Methode für in vivo Experimente ist unter Bearbeitung.Abstract Highly reactive hydroxyl radicals (hROS) 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 are 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 500myM) 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. 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 (1-12) and in vivo (13-16) 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). The dialysis probes were perfused at flow-rate of 3 myl/min with artificial cerebrospinal fluid CSF containing 250 mM TA2-. The dialysis samples were collected every 20 min, for a total detection time of approximately four hours. Calibration was absolutely necessary in order to obtain reliable data from on line experiments and to confirm the accuracy of the results. In order to validate this method, for each rat data from the on line monitoring, were compared with those obtained from the HPLC analysis. Therefore, the dialysis samples collected during on line experiments were analyzed for OH-TA and amino acids with HPLC with fluorimetric detection, using ex = 340 nm and em = 455 nm, for the amino acid OPA-derivatives, and ex = 315 nm and em = 435 nm, for OH-TA. Mobile phase consisting of methanol and potassium acetate (0.1 M, pH adjusted to 5.48 with glacial acetic acid) at a flow rate of 0.9 ml/min in a 3 linear step gradient (from 25% to 90% methanol) was used. The on line results were in good agreement with those obtained by HPLC. Further on a new in vitro HPLC-method based on the complexation of Fe(II) with bathophenanthroline disulphonate (BA) was developed using commonly UV/VIS detector at selective UV/VIS band 535 nm. For gradient elution a mobile phase containing 10 mM HEPES buffer adjusted to pH 7.5 and a flow rate of 20 myl/min was utilised. The objective of this study was to develop and evaluate a rapid method for iron determination with salt of sulfonated bathophenanthroline allowing an application in microdialysis experiments Essentially this on line method allows a near real time monitoring of hROS, as the fluorescence of 2-hydoxyl therephthalic acid (OH-TA) is highly selective. 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

Synthesis, characterization, antioxidant, cytotoxic, and DNA cleavage studies of ruthenium(III) complexes containing N-substituted thiosemicarbazone/semicarbazone

The synthesis, characterization, and biological studies of ruthenium(III) complexes containing ONS/ONO tridentate ligands are reported. Reactions of 1,2-naphthoquinone N-substituted thiosemicarbazone/semicarbazone (HLn; n = 1-4) with [RuCl3(EPh3)3] (E = P or As) gave [RuCl2(EPh3)(Ln)], which were characterized by elemental analysis, FT-IR, UV-Vis, magnetic susceptibility measurement, ESR spectra, ESI-MS, electrochemical studies, and powder X-ray diffraction pattern. The free radical scavenging ability, assessed by antioxidant assays involving DPPH and hydroxyl radicals, showed that the complexes possess excellent radical scavenging properties. The in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed that the ruthenium(III) complexes exhibited effective cytotoxic activity against selected cancer cells. Furthermore, the complexes were found to promote the cleavage of plasmid pBR322 DNA

The Application of Bathophenanthroline for the Determination of Free Iron in parallel with hROS in Microdialysis Samples

Free, non-protein bound, Fe(II), which can catalyse the formation of the toxic　highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from　microdialysis experiments has been limited by the small amounts of sample available.　New method. This work describes the development of a HPLC, with absorbance detection,　method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS),　which allows a complete extracellular iron analysis with the small sample amounts that are　available from in vivo microdialysis in rat brain.　Results. Microdialysis experiments using 6-hydroxydopamine stimulation, showed that basalas　well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel　with measurements of hROS formation.　Comparison with existing methods. Although a spectrophotometric BS-based assay has been　reported for use in microdialysis samples from large animals, the present procedure is　applicable to the small sample sizes available from studies in rat brain. It is simpler than the　alternative, involving inductively-coupled plasma mass spectrometry.　Conclusions. The procedure described is simple and sensitive, giving a linear response in the　Fe(II) concentration range of 50 -2000 nM. A 20 min microdialysis sample (flow-rate 3　μl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II)　and total iron whilst leaving sufficient sample volume for determining hROS and amine or　amino-acid neurotransmitter release.

In Vitro DNA/BSA Binding, Anticancer and Normal Cell Activity of Pd(II) Complexes: Substitution Behaviour and Computational Study

sulfur containing bio-molecules have been carried out under pseudo first order condition. In addition, theoretical investigation has also been considered for structural optimisation, frontier molecular orbital mapping, TD-DFT and NBO analysis. To get an insight into the cytotoxicity, the complexes have been treated on three different cancer cell lines (A549, HeLa, Hep G2) which reveal comparable anticancer activity of the reported complexes with the clinically acquainted anticancer drug Cisplatin. Very less complex initiated reactive oxygen species (ROS) with low degree of cell death in two different non-tumour cell lines (L6 myotubes and HEK 293) indicate minimum normal cell toxicity of the complexes. sulfur containing bio-molecules have been carried out under pseudo first order condition. In addition, theoretical investigation has also been considered for structural optimisation, frontier molecular orbital mapping, TD-DFT and NBO analysis. To get an insight into the cytotoxicity, the complexes have been treated on three different cancer cell lines (A549, HeLa, Hep G2) which reveal comparable anticancer activity of the reported complexes with the clinically acquainted anticancer drug Cisplatin. Very less complex initiated reactive oxygen species (ROS) with low degree of cell death in two different non-tumour cell lines (L6 myotubes and HEK 293) indicate minimum normal cell toxicity of the complexes

Continous Determinaton of hROS during Microdialysis

A procedure has been developed for the direct determination of hROS formation by monitoring the conversion of terephthalic acid to, the highly fluorescent, 2-hydroxy terephthalate by direct flow fluorimetry. The method should allow the rapid assessment of hROS formation evoked by a variety of neurotoxins and other compounds in microdialysis experiments in vivo