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

Abstract

The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 μmol/l) or salicylic acid (100 μmol/l) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25–40 μmol/l) 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 μmol/l, flow rate 2 μl/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.