Scavenging of reactive species by antioxidants: How to assess antioxidant activity

Chemico-Biological Interactions 169 (2007) 132–143

Abstracts of the Dutch toxicology days, 12th–13th June 2007

Scavenging of reactive species by antioxidants: How to assess antioxidant activity

knowledge of the in vivo situation and a careful design of the in vitro assay are necessary.

J.M. Balk, G.R.M.M. Haenen, A. Bast

doi:10.1016/j.cbi.2007.06.005

Department of Pharmacology and Toxicology, Maastricht University, Maastricht, The Netherlands During many processes in the human body, reactive species are formed. Antioxidants, produces by the human body or available through the diet, can protect human cells against the damage caused by these reactive species. The quest for the most efficient antioxidant is ongoing as countless compounds are screened for their antioxidant activity in vitro by competition assays. Unfortunately, the results of the different competition assays are not in consensus for the ranking of antioxidants according to their activity. We found that the antioxidant activity, described by its EC50 , is dependent on • • • •

the reaction rate constant of the detector, the reaction rate constant of the antioxidant under test, the concentration of reactive species used, the stoichiometry of the reaction of the antioxidant with the reactive species, and • the total capacity of the antioxidant. We also found that there are two kinds of antioxidant activity assays: the competition assay and the capacity assay. In the competition assay, the reaction rate constant of the antioxidant is the discriminating factor for the antioxidant activity. In the capacity assay, the capacity of the antioxidant determines the antioxidant activity. A simple test system is developed to indicate the discriminating factor in the assay used. To give an adequate description of the antioxidant activity, detailed 0009-2797/$ – see front matter doi:10.1016/j.cbi.2007.06.004

Modulating hesperetin bioavailability at the level of its intestinal metabolism and ABC transporter mediated efflux studied in Caco-2 monolayers W. Brand a,b , P.A.I. van der Wel a , G. Williamson b , P.J. van Bladeren a,b , I.M.C.M. Rietjens a a

Division of Toxicology, Wageningen University, Wageningen, The Netherlands b Nestl´ e Research Centre, Lausanne, Switzerland Biotransformation and transport from intestinal cells back into the intestinal lumen by ATP binding cassette (ABC) transporters, located in the apical membrane of intestinal cells, limit the bioavailability of many biofunctional ingredients. Such ingredients include hesperetin, a citrus flavonoid, which is of interest because of its proposed beneficial health effects. We studied the metabolism and transport of hesperetin in vitro using a two-compartment transwell system with Caco2 cell monolayers, grown on a microporous membrane dividing an apical and basolateral compartment, simulating the lumen and blood/plasma side of the intestinal transport barrier. We exposed the Caco-2 monolayers apically to hesperetin and investigated the formation and transport of hesperetin conjugates. Our results show that un-conjugated hesperetin appeared at the basolateral side of the Caco-2 monolayers. Results show that hesperetin was also metabolized extensively into two major metabolites, the dominant one being hesperetin7-O-glucuronide, which was identified by UPLC-MS in combination with 1 H-NMR. Both metabolites were