Hemoglobin-dependent differences in the antioxidant properties of uric acid and albumin: an in vitro analysis

Abstract

Oxidative stress is a major contributor to morbidity in patients with end-stage kidney disease. The antioxidant properties of vitamin E-coated hemodialyzers have attracted increasing attention. However, the intrinsic antioxidant capacity of dialysis-related substances themselves remains unclear despite their potential relevance to redox balance during hemodialysis. In this study, we aimed to clarify the antioxidant behavior of several clinically relevant compounds, including uric acid, ascorbic acid, glutathione, glycerin, and recombinant human albumin, by evaluating their ability to suppress chemiluminescence generated by Fenton’s reagent (FeSO4 and H2O2) in aqueous media. To assess the influence of hemoglobin, each antioxidant was dissolved in distilled water or a 3 μM human hemoglobin solution. Additionally, recombinant human albumin was tested in the presence of 1, 2, and 30 μM hemoglobin. A 50 μL sample was mixed with 100 μL of Fenton’s reagent, and luminescence intensity was quantified using a luminometer. The concentrations of FeSO4 and H2O2 were 1 and 10 mM, respectively. The half-maximum inhibitory concentration (IC50) and its negative logarithm (pIC50) were calculated from sigmoidal dose–response curves, and relative activities were compared with those of glycerin, a known hydroxyl radical scavenger. In distilled water, glutathione, ascorbic acid, and uric acid exhibited approximately 100‑, 1000‑, and 20,000‑fold higher antioxidant activities, respectively, than glycerin. Although uric acid substantially suppressed chemiluminescence in distilled water, it showed no antioxidant effect in hemoglobin-containing solution. In contrast, recombinant human albumin increased luminescence intensity when tested alone but markedly suppressed luminescence in the presence of hemoglobin. Based on pIC50 values, antioxidant capacity in distilled water decreased in the order uric acid > ascorbic acid > glutathione > glycerin. Albumin showed clear antioxidant activity under hemoglobin-containing conditions, most likely attributable to its heme-binding ability. Uric acid, ascorbic acid, and glutathione exhibited strong antioxidant activity against Fenton’s reagent in aqueous systems, with uric acid showing the highest potency. Furthermore, recombinant human albumin showed antioxidant activity, most likely through heme sequestration. These findings provide mechanistic insights into the strongly context-dependent antioxidant behavior of dialysis-related solutes. Further validation in ex vivo and clinical studies is required.