Dataset Information
A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin.
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ABSTRACT: Objectives
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.Design and methods
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%.Results
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.Conclusions
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.
SUBMITTER: Tada S
PROVIDER: S-EPMC9192801 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
Publications
A simple colorimetric assay to determine the concentration and proportion of human mercaptalbumin.
Practical laboratory medicine 20220523
<h4>Objectives</h4>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.<h4>Design and methods</h4>Michler's Hydrol (4,4'-Bis(dimethylamino)benzhydrol) is a blue dye with a maximum abso ...[more]