title: Development of a new assay for the screening of hypochlorous acid scavengers based on reversed-phase high-performance liquid chromatography.
abstract: A new assay for the screening of hypochlorite/hypochlorous acid (XOCl) scavengers, based on the reversed-phase high performance liquid chromatographic analysis of human serum albumin (HSA, 0.2% in 100 mM sodium phosphate, pH 7), before and after oxidation by XOCl (1.6 mM), was developed. XOCl induced a significant decrease of the area under the chromatographic peak of HSA at 280 nm due to the oxidation of the aromatic amino acids tryptophan and tyrosine, as suggested by the literature and by the chromatographic analyses and the electrochemical study performed here. The assay was validated by testing known XOCl scavengers such as ascorbic acid, cysteine, glutathione, S-methylglutathione and alpha-lipoic acid and other antioxidants such as carnosine and chlorogenic acid, which inhibited the oxidation of HSA. Quantitative activities were calculated using an original formula based on the changes of the area of the albumin peak. Electrochemical data collected here in a homogeneous medium showed that the anodic potentials of the antioxidants tested are less positive (ascorbic acid, chlorogenic acid and cysteine) or similar (alpha-lipoic acid) compared with those of the aromatic residues (tryptophan and tyrosine) of HSA oxidized by XOCl. However, as expected, carnosine, glutathione and S-methylglutathione were inactive at a glassy-carbon, gold or platinum electrode.
authors.affiliation: Dipartimento di Farmacologia delle Sostanze Naturali e Fisiologia Generale Università di Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Gatto
authors.forename: Omidreza
authors.initials: O
authors.lastname: Firuzi
authors.forename: Roberta
authors.initials: R
authors.lastname: Agostino
authors.forename: Eleonora
authors.initials: E
authors.lastname: Grippa
authors.forename: Angela
authors.initials: A
authors.lastname: Borsò
authors.forename: Francesca
authors.initials: F
authors.lastname: Spinelli
authors.forename: Lucia
authors.initials: L
authors.lastname: Pavan
authors.forename: Marzia
authors.initials: M
authors.lastname: Petrolati
authors.forename: Rita
authors.initials: R
authors.lastname: Petrucci
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Marrosu
authors.forename: Luciano
authors.initials: L
authors.lastname: Saso
mesh_terms: D000975:Antioxidants / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D006997:Hypochlorous Acid / Q000737:chemistry*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry
chemical_list: D000975:Antioxidants; D012709:Serum Albumin; D006997:Hypochlorous Acid