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). 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 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 making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO (rate constant ~8.7×106M-1s-1) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA ~5mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1-200μM. We found that the rate constant for LA+HO is diffusion-controlled (~1×1010M-1s-1in water at 25°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.
Dipartimento di Scienze della Salute, Università degli Studi di Milano, c/o Ospedale S. Paolo, Via A. di Rudinì, 8-20142 Milano, Italy.
Andrea
A
Brizzolari
Dipartimento di Scienze della Salute, Università degli Studi di Milano, c/o Ospedale S. Paolo, Via A. di Rudinì, 8-20142 Milano, Italy.
Giuseppe M
GM
Campisi
Dipartimento di Scienze della Salute, Università degli Studi di Milano, c/o Ospedale S. Paolo, Via A. di Rudinì, 8-20142 Milano, Italy.
Enzo
E
Santaniello
Department of Medicinal Chemistry and Drugs & Advanced Sciences Research Center, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran.
Nima
N
Razzaghi-Asl
Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University of Rome, P. le Aldo Moro, 5-00185 Rome, Italy.
Luciano
L
Saso
Istituto di Chimica Biomolecolare del CNR, via Paolo Gaifami 18, 95126 Catania, Italy. Electronic address: mario.foti@cnr.it.
Mario C
MC
Foti
D000097:Acetonitriles; D003855:Deoxyribose / Q000737:chemistry*; D016166:Free Radical Scavengers / Q000737:chemistry*; D017665:Hydroxyl Radical / Q000737:chemistry*; D009930:Organic Chemicals / Q000737:chemistry; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D008063:Thioctic Acid / Q000737:chemistry*
D000097:Acetonitriles; D016166:Free Radical Scavengers; D009930:Organic Chemicals; D012997:Solvents; D017665:Hydroxyl Radical; D003855:Deoxyribose; D008063:Thioctic Acid; C032159:acetonitrile
2-Deoxyribose degradation assay; Acetonitrile; Hydroxyl radical; Lipoic acid; Scavenging activity