Salicylate hydroxylation products assay as a marker of oxidative stress in man: A sensitive HPLC-electrochemical method

Summary

The in vivo measurement of highly reactive free radicals, such as ^.OH radical, in humans is very difficult. For this reason, secondary products of oxidative stress are frequently measured. The most indirect methods (TBARs [thiobarbituric acid reactants] test, conjugated diene, hydroperoxides) are rather unspecific and can give conflicting results. New, more specific markers are currently under investigation (protein oxidation, DNA adducts and aromatic probes). All these methods are based on the ability of ^.OH to attack the benzene rings of aromatic molecules to produce hydroxplated compounds that can be measured directly.

In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives 2,3- and 2,5-dihydroxybenzoic acid [2,3- and 2,5-DHBA]). The latter can also be produced by enzymatic pathways through the cytochrome P-450 system, while the former is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-DHBA. following oral administration of salicylate in its acetylated form (aspirin), could be proposed for assessment of oxidative stress in vivo.

In this work, evidence is presented for a sensitive method for the detection of hydroxyl free radical generation in vivo. 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 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.