A novel ¹⁹F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution ¹⁹F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4- trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by ¹⁹F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)- trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, k(F), that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. k(F) values obtained for low- molecular-mass compounds fall in the range 0.17 < k(F) < 1.5 and are in good agreement with second order rate constants (k₂(OH)) for the reaction of the antioxidant with hydroxyl radicals. The k(F) value for serum albumin is much larger (46.9) than that predicted from the reported k₂(OH) value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.