Antioxidant Capacity of Reduced and Hydrogen-Peroxide-Oxidized Human Serum Albumin and Its Modulation by Hydroxychloroquine and Tigecycline

Abstract

BACKGROUND. Oxidative stress has long been implicated in the development of pathological conditions, and direct evidence of in vivo oxidative stress exists in oxidative stress-related diseases. Proteins are highly susceptible to oxidative stress, but studies of interactions among pharmacological agents, biologically active substances, and oxidatively modified proteins have been limited. The antioxidant capacity of HSA plays multiple key roles as a sensor and regulator of signal transduction and of drug efficacy in response to H₂O₂. OBJECTIVES. The objectives of the study were to compare the antioxidant capacity of the reduced and H₂O₂-treated HSA and evaluate the dependence of hydroxychloroquine and tigecycline on the physiologically important function of HSA from its initial redox state. METHODS. Human serum albumin (HSA) fraction V, a free fatty acid, was obtained from MP Biomedicals, LLC and then reduced with dithiothreitol (DTT), 10 mM, or oxidized by 10 mM hydroperoxide. The antioxidant capacity of HSA was studied in denaturing conditions using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and measured by the UV-vis absorption spectrum maximum at 517 nm, and the native antioxidant capacity (AC) by converting the reaction of the cationic radical 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and the absorbance intensity at 734 nm. SPSS v.22.0 was used for statistical analysis of the data, and results are presented as mean ± standard deviation. Differences are considered significant when p<0.05. RESULTS. H₂O₂-induced oxidation of HSA leads to a decrease in the AC of the protein, and after 3 min, it reaches only 43-46% of that observed for reduced HSA. Tigecycline (TGC, 2×10-4 M) increased the antioxidant potential of HSA, scavenging ABTS in native conditions, by 55.3% after 15 min of incubation, and that of the HCQ (2×10-4 M)-HSA complex by 43%. H₂O₂- treated HSA lost the AC more than 50% at all times of the experiments, and after incubation with HCQ and TGC, the AC reached 49% and 69.6% for reduced HSA. There were no significant differences between the AC of the reduced and oxidized forms of HSA under denaturing conditions (DPPH assay); only after 45 min did the changes become significant following the addition of free radicals. CONCLUSIONS. The present findings provide insights into the relationship between the oxidative status of HSA, characteristic of oxidative-based diseases, and its functional deterioration in blood. H₂O₂-treated HSA showed decreased AC under native conditions in the ABTS assay. At the same time, HCQ and TG can restore AC in oxidatively modified HSA, and these changes do not appear when measuring AC under HSA denaturation conditions. This study may help elucidate the binding mechanism of drugs to serum proteins, providing additional rationale for their use as anti-inflammatory and antimalarial agents.