Synergistic interactions of antioxidant nutrients in a biological model system

Abstract Objective The antioxidant activity of fat- and water-soluble antioxidant nutrients and their interactions in physiologic concentrations were determined in an in vitro biological model system. Methods Reconstituted human serum consisting of delipidized human serum (DHS) combined with phosphatidylcholine liposomes (PCL) was used to determine antioxidant activities of physiologic concentrations of antioxidant nutrients. Radicals were initiated with 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (2 mmol/L), and oxidation was monitored by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid. Fat-soluble antioxidant nutrients were incorporated into the PCL prepared by sonication and suspended in DHS to avoid any interference by the endogenous fat-soluble antioxidants. Water-soluble antioxidants were added directly into the DHS. The oxidation kinetics were monitored every 5 min up to 210 min using a microplate reader (excitation wavelength 500 nm, emission wavelength 520 nm). Results We confirmed the synergistic protective effect of the combination of ascorbic acid (1–5 μM) and α-tocopherol (1–5 μM) against the oxidation of DHS with PCL. Furthermore, physiologic concentrations of 1) β-carotene (0.1, 0.5 μM) and α-tocopherol (2.5, 5.0 μM), 2) β-carotene (0.1, 0.5 μM) and ascorbic acid (2.5 μM), and 3) uric acid (10 uM) and α-tocopherol (2.5, 5.0 μM) synergistically protected oxidation of reconstituted human serum. Conclusion The present study results suggest a wide antioxidant network between water- and fat-soluble antioxidant nutrients in a biological system, although their actions in vivo warrant further study.