Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals

C. Themann, Peter Teismann, K. Kuschinsky, B. Ferger

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Abstract

The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 mu mol/1) or salicylic acid (100 mu mol/1) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25-40 mu mol/1) of equimolar hydrogen peroxide and ferrous ions. Both, phenylalanine and salicylic acid were able to trap hydroxyl radicals in a reliable way indicated by the linear relationship between the concentration of the Fenton reagents and either the phenylalanine derived products (ortho-, meta-, para-tyrosine) or the salicylic acid-derived products (2,3- and 2,5-dihydroxybenzoic acid (DHBA)). In vivo experiment: Wistar rats were implanted with microdialysis probes and striatal perfusion with either 5 mmol/l phenylalanine or 5 mmol/l salicylic acid was performed. Addition of the dopaminergic neurotoxin 6-hydroxydopamine (100 mu mol/l, flow rate 2 mul/min, 60 min) to the perfusion fluid significantly increased the concentrations of ortho- and meta-tyrosine or 2,3-DHBA in comparison to control animals. All increases determined were rapidly reversible after changing back to pre-stimulation conditions. The results demonstrate that aromatic hydroxylation of phenylalanine or salicylic acid is a useful technique to investigate hydroxyl free radical formation in vitro and in vivo. Advantages and disadvantages of both methods are discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Original languageEnglish
Pages (from-to)57-64
Number of pages7
JournalJournal of Neuroscience Methods
Volume108
Issue number1
Early online date11 Jul 2001
DOIs
Publication statusPublished - 15 Jul 2001

Keywords

  • hydroxyl radicals
  • phenylalanine
  • salicylic acid
  • 6-hydroxydopamine
  • microdialysis
  • HPLC
  • rat striatum
  • in-vivo
  • reperfusion injury
  • salicylate
  • dopamine
  • generation
  • ischemia
  • brain

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