HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROCHEMICAL DETERMINATION OF SALICYLATE HYDROXYLATION PRODUCTS AS AN IN-VIVO MARKER OF OXIDATIVESTRESS

The in vivo measurement of highly reactive free radicals, such as hydr oxyl radical ((OH)-O-.), in humans is very difficult if not impossible . Specific markers are currently under investigation (amino acid hydro xylatin, protein, DNA adducts, and aromatic probes). They are based on the ability of (OH)-O-. to attack aromatic molecules to produce hydro xylated compounds that can be measured directly. In vivo, radical meta bolism of salicylic acid produces two main hydroxylated derivatives, i .e., 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA). The measu rement of 2,3-DHBA, following oral administration of salicylate or its acetylated form (aspirin), has been proposed for assessment of in viv o oxidative stress. In this work, a sensitive method for the detection of in vivo (OH)-O-. generation is presented. The methodology employs a high-pressure liquid chromatography with electrochemical detection f or the identification and quantification of the hydroxylation products from the reaction of (OH)-O-. with salicylate. A detection limit of l ess than 0.1 pmol for the hydroxylation products has been achieved wit h electrochemical detector responses which were linear over at least f ive orders of magnitude. Using this technique, we measured plasma leve ls of 2,3- and 2,5-DHBA and dihydroxylated derivatives/salicylic acid ratios following the administration of 1000 mg aspirin in 20 healthy s ubjects. In the same individuals, plasma levels of thiobarbituric acid reactants (TBARs), a major index of lipid peroxidation, were also mea sured and correlation with hydroxylated products was sought. The plasm a level of TBARs was positively correlated with the 2,5-DHBA/salicylic acid ratio, but not with the absolute plasma level of 2,3-DHBA. (C) 1 995 Academic Press, Inc.