A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin

• Published in: Practical laboratory medicine 2022-08, Vol.31, p.e00281-e00281, Article e00281
• Main Authors: Tada, Sawaki, Yasukawa, Keiko, Yatomi, Yutaka, Uchiki, Tomoaki
• Format: Article
• Language: English
• Subjects: 4,4′-bis(dimethylamino)benzhydrol; Albumin redox states; Cys34; Cysteine 34; HMA; HNA; Human mercaptalbumin; Human non-mercaptalbumin; Human serum albumin; Michler's hydrol; Original; Oxidative stress; Oxidized albumin; Reduced albumin
• ISSN: 2352-5517; 2352-5517
• DOI: 10.1016/j.plabm.2022.e00281

Human serum albumin can take on two forms, mercaptalbumin (HMA) or non-mercaptalbumin (HNA), depending on the redox status of its Cys34. The ratio of HMA and HNA is considered to be a novel biomarker of oxidative stress. While HPLC and mass spectrometry are established methods to measure HMA and HNA, a simple colorimetric assay was applied to measure this biomarker. Michler's Hydrol (4,4′-Bis(dimethylamino)benzhydrol) is a blue dye with a maximum absorption at 612 nm, and its absorption decreases when it reacts with a thiol group. Concentrations of HMA in serum samples from 36 healthy subjects were measured based on absorption changes of Michler's Hydrol. The proportion of HMA (HMA%) in total albumin was also obtained by dividing the HMA concentration by total albumin concentration, which was obtained by a bromocresol purple (BCP) assay. The proportion of HNA (HNA%) was obtained by subtracting HMA% from 100%. HMA concentrations obtained by Michler's Hydrol assay were highly correlated (r2 = 0.97) with reference values obtained by HPLC (HMA%) and BCP assay (total albumin). The HNA% obtained by Michler's Hydrol and BCP assays combined also gave a good correlation (r2 = 0.96) and a small deviation (average 2.4%) with respect to HPLC as a reference method. A colorimetric assay using Michler's Hydrol was optimized for a 96-well plate format so that it can be easily performed in a standard laboratory setting. This assay gives HMA concentrations and HNA proportions comparable to HPLC. [Display omitted] •Concentrations of the reduced form of human serum albumin (human mercaptalbumin) were measured by a colorimetric assay using Michler's Hydrol.•The assay uses the total thiol concentration to approximate the human mercaptalbumin concentration.•Accuracy was improved by alkylating part of the samples and canceling the contribution of the non-thiol component to the colorimetric changes.•Combining Michler's Hydrol assay with bromocresol purple assay enabled us to obtain the proportions of mercaptalbumin and non-mercaptalbumin, which correlated with HPLC as a reference method.