A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices.

INTRODUCTION: Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well.
METHODS: In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. RESULTS AND DISCUSSION: The ECL method is fast (10min) with an interassay precision of <10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r=0.84, 95% confidence interval, CI=0.78-0.89, P<0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p<0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p<0.01). Pearson's statistics showed the strongest positive correlation with serum uric acid (r=0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.