A unique two-way approach for the validation of total antioxidant capacity of serum samples.

The human body is constantly exposed to a large variety of reactive oxygen species that are implicated in many pathophysiological conditions (atherosclerosis, cancer, neurodegenerative diseases etc.). Monitoring the antioxidant status of biological fluids could be used as an early warning sign 'biomarker' of possible disease onset. However, although several methods have been developed, questionable sensitivity, unreliability and non-reproducibility hamper all, making it difficult to have an internationally accepted standardized method. This study presents and demonstrates the remarkable ability of a newly developed antioxidant capacity assay method based on tetramethoxy azobismethylene quinone (TMAMQ) to measure the total antioxidant capacity of serum samples using three complimentary approaches. Using an UV-Vis spectroscopy and oxygen sensor, the reduction of TMAMQ by serum antioxidants was compared to either the formation of syringaldazine or consumption of oxygen. After adding a fraction of human serum, 4·01 μM TMAMQ was reduced to syringaldazine from a stock of 11·74 μM TMAMQ. Subsequent addition of laccase resulted in the oxidation of the formed syringaldazine back to TMAMQ resulting in an increase in TMAMQ concentration to 11·71 μM (re-establishing almost the same initial concentration of TMAMQ) while consuming 1·04 μM molecular oxygen. The reduction of TMAMQ by serum samples is directly proportional to the consumption of oxygen and the formation of syringaldazine. This means that either the formation of syringaldazine or oxygen consumption can be used to validate or confirm data obtained through monitoring TMAMQ reduction.