High-performance liquid chromatography-electrochemical determination of salicylate hydroxylation products as an in vivo marker of oxidative stress.

The in vivo measurement of highly reactive free radicals, such as hydroxyl radical (.OH), in humans is very difficult if not impossible. Specific markers are currently under investigation (amino acid hydroxylatin, protein, DNA adducts, and aromatic probes). They are based on the ability of .OH to attack aromatic molecules to produce hydroxylated compounds that can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives, i.e., 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA). The measurement of 2,3-DHBA, following oral administration of salicylate or its acetylated form (aspirin), has been proposed for assessment of in vivo oxidative stress. In this work, a sensitive method for the detection of in vivo .OH generation is presented. The methodology employs a high-pressure liquid chromatography with electrochemical detection for the identification and quantification of the hydroxylation products from the reaction of .OH with salicylate. A detection limit of less than 0.1 pmol for the hydroxylation products has been achieved with electrochemical detector responses which were linear over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA and dihydroxylated derivatives/salicylic acid ratios following the administration of 1000 mg aspirin in 20 healthy subjects. In the same individuals, plasma levels of thiobarbituric acid reactants (TBARs), a major index of lipid peroxidation, were also measured and correlation with hydroxylated products was sought. The plasma 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.