Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals.
The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 micromol/l) or salicylic acid (100 micropmol/l) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25--40 micromol/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 micromol/l, flow rate 2 microl/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.
Themann
C
C
Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Ketzerbach 63, D-35032 Marburg, Germany.
Teismann
P
P
Kuschinsky
K
K
Ferger
B
B
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D005841:Gentisates / Q000378:metabolism; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism*; D007536:Isomerism; D008297:Male; D017551:Microdialysis / Q000379:methods*; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D016627:Oxidopamine / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020156:Salicylic Acid / Q000378:metabolism / Q000494:pharmacology; D013565:Sympatholytics / Q000494:pharmacology
D005841:Gentisates; D006841:Hydrocarbons, Aromatic; D013565:Sympatholytics; D017665:Hydroxyl Radical; D010649:Phenylalanine; D016627:Oxidopamine; D020156:Salicylic Acid