Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis.
Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks 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 as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/muL. Derivatization of samples 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. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate 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 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2- forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.
Freinbichler
Wolfhardt
W
Institute for Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria.
Colivicchi
Maria A
MA
Fattori
Manuela
M
Ballini
Chiara
C
Tipton
Keith F
KF
Linert
Wolfgang
W
Della Corte
Laura
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D017665:Hydroxyl Radical / Q000032:analysis / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D007608:Kainic Acid / Q000494:pharmacology; D008297:Male; D017551:Microdialysis; D009448:Neurochemistry / Q000379:methods*; D009498:Neurotoxins / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000032:analysis / Q000378:metabolism*; D010795:Phthalic Acids / Q000138:chemical synthesis / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D009764:o-Phthalaldehyde / Q000737:chemistry
D000596:Amino Acids; D009498:Neurotoxins; D018377:Neurotransmitter Agents; D010795:Phthalic Acids; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical; C406866:2-hydroxyterephthalic acid; D009764:o-Phthalaldehyde; C011363:terephthalic acid; D007501:Iron; D007608:Kainic Acid