[Zurück]

B. Misini:
"Development of an Online Fluorescence Method for near real time in vivo Monitoring of Hydroxyl Radicals in Rats";
Betreuer/in(nen), Begutachter/in(nen): W. Linert, L. Della Corte; Institut für angewandte Synthesechemie, 2012; Rigorosum: 06.11.2012.

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 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.

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 μl/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 λex = 340 nm and λem = 455 nm, for the amino acid OPA-derivatives, and λex = 315 nm and λem = 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 µl/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.

in vivo measurements, highly reactive oxidative species