Rapid and convenient detection of ascorbic acid using a fluorescent nitroxide switch

Ascorbic acid is a small-molecule reductant with multiple functions in vivo. Reducing ascorbic acid intake leads to a lack of hydroxylation of prolines and lysines, causing a looser triple helix and resulting in scurvy. Ascorbic acid also acts as an antioxidant to prevent oxidative stress. Because ascorbic acid is related to disease states, rapid and convenient detection of ascorbic acid should be useful in diagnosis. Nitroxide is reduced to the corresponding hydroxylamine by ascorbic acid and a sensitive and novel approach to its detection employs covalent coupling of nitroxide with a fluorophore, leading to intramolecular quenching of fluorescence emission by electron-exchange interactions. Here, we developed a new fluorophore-nitroxide probe, Naph-DiPy nitroxide, for ascorbic acid. Naph-DiPy nitroxide rapidly reacted with ascorbic acid and showed fluorescence enhancement, but not in response to other reductants or reactive oxygen species. To confirm the practical usefulness of the fluorophore-nitroxide probe, we demonstrated the use of Naph-DiPy nitroxide for the measurement of ascorbic acid in the plasma of osteogenic disorder Shionogi rats when fed an ascorbic acid-deficient diet. The results suggest that this novel fluorophore-nitroxide probe could sensitively and easily detect ascorbic acid and be useful as a tool for the diagnosis of disease states.