A simple assay for the measurement of plasma antioxidant status using spontaneous autoxidation of homovanillic acid

Abstract

Introduction: Reactive oxygen species (ROS) contribute to the development of pathophysiological processes, hence the increasing interest in modulating the antioxidant status of patient by nutritional or pharmacological intervention. Antioxidants act by preventing the formation of ROS (inhibitory effect) and/or by trapping these species (scavenger effect). We have developed a simple, sensitive, and reliable test to measure the total antioxidative efficiency of plasma or other biological fluids using microliter samples.

Methods: Autoxidation of homovanillic acid (HVA) gives rise to fluorescent dimers. Antioxidants contained in the plasma (or free aqueous solutions) scavenge the ROS involved in this process and transiently stop the linear increase in fluorescence intensity during a time (delay) proportional to the total concentration of antioxidants and their scavenging efficiency. In addition to this scavenging effect, the kinetics of HVA autoxidation, restarting after the delay, reflects the ability of the plasma antioxidants to inhibit the ROS-triggered autoxidation.

Results: The rate of the HVA autoxidation depended on the temperature, the protonation of the phenolic group, and on the presence of peroxide, peroxyl radicals, and peroxidase as well as metal ions. This Fenton-like reaction was transiently stopped by various ROS scavengers including quercetin, ascorbic acid, and thiol derivatives (glutathione and N-acetylcystein) while metal chelating agents such as desferrioxamine, ethylene diamine tetracetic acid (EDTA), and polyamine only reduced its rate.

Discussion: The main advantages of this new assay are its versatility to investigate in a single run both the scavenging and inhibitory components of the antioxidant capacity, and its relevance to the reactive hydroxyl radical. As shown in this study, the increase in the antioxidant capacity of human plasma during pharmacological supplementation with antioxidant illustrates one of the various fields of application of this assay.