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

Background 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. Here, 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 (FeSO ₄  + H ₂ O ₂ ) in aqueous media. Methods 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 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 FeSO ₄ and H ₂ O ₂ were 1 and 10 mM, respectively. The half-maximal inhibitory concentration (IC ₅₀ ) and its negative logarithm (pIC ₅₀ ) were calculated using sigmoidal dose–response curves, and relative activities were compared with glycerin, a known hydroxyl radical scavenger. Results In distilled water, glutathione, ascorbic acid, and uric acid exhibited approximately 100‑, 1,000‑, and 25,000‑fold higher antioxidant activities than glycerin, respectively. 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 pIC ₅₀ values, the antioxidant capacities in distilled water ranked from high to low as: uric acid > ascorbic acid > glutathione > glycerin. Albumin demonstrated clear antioxidant activity under hemoglobin-containing conditions, primarily attributable to its heme-binding ability. Conclusion Uric acid, ascorbic acid, and glutathione exhibited strong antioxidant activity against Fenton’s reagent in aqueous systems, with uric acid showing the highest potency. Recombinant human albumin also demonstrated antioxidant activity through heme sequestration, yielding a pIC ₅₀ higher than those of glutathione and glycerin. These findings highlight the context-dependent nature of antioxidant behavior among dialysis-related solutes and provide mechanistic insight into oxidative stress during hemodialysis, particularly in settings of microhemolysis and free heme accumulation. These findings may contribute to the development of strategies aimed at improving redox balance in patients undergoing renal replacement therapy.