Determination of salicylate hydroxylation products as an in vivo oxidativestress marker

The in vivo measurement of highly reactive free radicals, such as the (OH)- O-. radical, is very difficult. New specific markers, which are based on th e ability of (OH)-O-. to attack the benzene rings of aromatic molecules, ar e currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). Th e latter acid can be also produced by enzymatic pathways through the cytoch rome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-DHBA, follow ing oral administration of the drug acetyl salicylate, could be proposed fo r assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from t he reaction of (OH)-O-. with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system . A detection limit of < 1 pmol for the hydroxylation products has been ach ieved with linear response over at least five orders of magnitude. Using th is technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following adminis tration of 1 g acetyl salicylate in 20 healthy subjects. (C) 2000 Elsevier Science Inc.