Hydrogen peroxide, antioxidant compounds and biological targets: an in vitro approach for determination of scavenging capacity using fluorimetric multisyringe flow injection analysis

Abstract

In the present work, an automatic method based on multisyringe flow injection analysis (MSFIA) was developed for determination of scavenging capacity against H(2)O(2) using a non-enzymatic fluorimetric assay for H(2)O(2) detection based on the formation of europium-tetracycline-H(2)O(2) complex. The MSFIA-fluorimetric methodology was developed to perform in-line the scavenging reaction of H(2)O(2) prior to reaction of the remaining H(2)O(2) with the fluorescent probe, using conditions close to those found in vivo regarding pH (6.9), temperature (37 degrees C) and H(2)O(2) concentration (25microM). This approach allowed the evaluation of scavenging capacity against H(2)O(2) in a non-competitive (antioxidant+H(2)O(2)) or a competitive (antioxidant+H(2)O(2)+biological target) scheme. Using the first strategy, IC(50) values determined for the antioxidant compounds glutathione reduced (1191+/-46microM) and pyruvate (446+/-49microM) were lower than those obtained for biological targets such as cysteine (2616+/-182microM), taurine (359+/-38mM) and adenine (2224+/-214microM), indicating that reactivity towards H(2)O(2) was higher for antioxidant compounds than for biological targets. However, when a competitive scheme was applied, the scavenging effectiveness against H(2)O(2) depended on the biological molecule present, showing that antioxidant assessment should also take into consideration the concomitant reactivity of biological molecules or structures that are prone to oxidative damage.