A novel method for measuring serum albumin redox state using a thiol-binding reagent

Background and objectives: Albumin is the most abundant serum protein, comprising a mixture of human mercaptalbumin (HMA, reduced albumin) and nonmercaptalbumin (HNA, oxidized albumin), depending on the redox state of the cysteine residue at position 34. The redox state of serum albumin has been viewed as an oxidative stress biomarker, and recent studies have found its potential as a protein nutrition biomarker (Tabata et al., Antioxidants 2021). Serum albumin redox state has normally been measured using HPLC. However, this requires an expensive equipment and laborious technique, and is not feasible in many clinical practices. To overcome these limitations, we developed a simple method to evaluate albumin redox state by separating HMA and HNA, using a thiol-binding reagent and detecting albumin fractions in colorimetric assays. Methods: HMA in serums of healthy adult volunteers (20 samples) were removed by reacting the free thiol group of HMA with a pyridyl disulfide-linked resin, e.g., a Cytiva Activated Thiol Sepharose 4B, or a Thermo Scientific EZ-Link HPDP-Biotin in combination with a Streptavidin Agarose Resin; HNA-containing fractions were then separated by ultrafiltration. Albumin concentrations in both serums (HMA+HNA) and the HNA-containing fractions were measured by a colorimetric method with bromocresol green (BCG) or bromocresol purple (BCP), and the ratio of HMA to total albumin (%HMA) was determined. The %HMA measured by the above method was then compared with the %HMA determined by a conventional HPLC method. Results: Removal of HMA from the serums, i.e., the purity of HNA in the HNA-containing fraction, was confirmed by HPLC. Simple linear regression analyses indicated that %HMA by this method and the one by HPLC were correlated significantly (r = 0.614-0.910). Conclusions: This novel and simple approach can be a valuable tool for measuring serum albumin redox state, a potential protein nutrition biomarker as well as an oxidative stress biomarker, in a variety of clinical settings.