Effect of organic co-solvents in the evaluation of the hydroxyl radical scavenging activity by the 2-deoxyribose degradation assay: The paradigmatic case of α-lipoic acid

The 2-deoxyribose degradation assay (2-DR assay) is an in vitro method broadly used for evaluating the scavenging activity against the hydroxyl radical (HO center dot). One of the major drawbacks of the assay, however, is that only water soluble compounds can be tested for their radical-scavenging activity. Lipoic acid (LA) is an excellent scavenger of HO center dot but it exhibits a low solubility in the aqueous milieu of the 2-DR assay and a high tendency to polymerize under a variety of conditions. We used LA as a paradigmatic substrate to evaluate the effect of several organic co-solvents in increasing its solubility. Most of these solvents, however, demonstrated to be potent scavengers of HO center dot making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO center dot (rate constant similar to 8.7 x 10(6) M-1 s(-1)) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA similar to 5 mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1-200 mu M. We found that the rate constant for LA + HO center dot is diffusion-controlled (similar to 1 x 10(10) M-1 s(-1) in water at 25 degrees C) and uninfluenced by the presence of small quantities of acetonitrile. Therefore, the use of acetonitrile in the 2-DR assay does not interfere with the test and may increase the solubility of the radical scavengers. (C) 2016 Elsevier B.V. All rights reserved.