Letter to the editor
Hydroxylation of salicylate as an assay for hydroxyl radicals: A cautionary note

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    For the measurement of extracellular hydroxyl radicals in the hippocampus, the implanted probe was perfused with artificial cerebrospinal fluid containing 10 mM salicylic acid, using a high-pressure pump (CMA/Microdialysis; RosLagsvägen, Stockholm, Sweden) at a flow rate of 1.2 μL/min [19]. It is known that salicylate can react with hydroxyl radicals to generate stable dihydroxybenzoic acid (DHBA) derivatives, in particular 2,3-DHBA, which can be used as an in vivo index of hydroxyl radical levels [20]. The volume of 2,3-DHBA in dialysates was measured by high-performance liquid chromatography using a two-channel electrochemical detector (LC-4C; Bioanalytical Systems, West Lafayette, IN).

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    Thereafter, sodium salicylate in Ringer's solution (0.5 nmol/μL per min) was perfused by a microinjection pump (Carnegie Medicine CMA/100, Stockholm, Sweden) to trap OH radicals [4,6] in the striatum, and basal levels of 2,3-dihydroxybenzoic acid (DHBA) were determined during a definite period. Brain dialysate (1 μL/min) was collected every 15 min in small tubes containing 15 μL of 0.1 N HClO4 to prevent amine oxidation and assayed immediately for 2,3-DHBA by high-performance liquid chromatography with electrochemical (HPLC-EC) procedure [6,13]. The recovery rate of 1 μM 2,3-DHBA was 12.0 ± 1.0% at a flow rate of 1 μL/min.

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