Introduction: In order to better understand the physiological and pathophysiological roles of reactive oxygen species (ROS), multiple blood and urine biomarkers of oxidative stress have been developed. The single free thiol (Cys34) in plasma albumin is a useful biomarker of oxidative stress because thiol groups are particularly sensitive to oxidation by ROS. The primary aim of this study was to develop a gel electrophoresis-based method (mPEG assay) that would be more widely accessible than existing chromatography techniques to assay the oxidation state of albumin Cys34.

Method: Blood samples were collected into a solution containing polyethylene glycol maleimide (malpeg). Plasma samples were divided into two aliquots, with a reducing agent added to one aliquot. Albumin bound to malpeg was separated from albumin by gel electrophoresis. The proportion of albumin in reduced form (–SH), disulphide form (–SSX) and irreversibly oxidised form (–SO₂, –SO₃) could then be calculated.

Results: Data for the mPEG assay was comparable to data from chromatographic and mass spectrometric assays. The mPEG assay was more sensitive than the albumin carbonyl assay for the detection of changes in albumin oxidation level in response to exposure to hydrogen peroxide or hypochlorous acid. This assay could also be performed on small blood samples (less than 10 µL) from fingerprick, thus facilitating longitudinal tracking of changes in albumin Cys34 oxidation level.

Conclusion: The mPEG assay is a user-friendly, highly sensitive, specific, cost-effective gel electrophoresis-based method for the assay of the oxidations state of albumin Cys34 as a biomarker of oxidative stress.

Highlights
• Protein thiol groups are sensitive to oxidation by reactive oxygen species.
• Plasma albumin contains a reduced cysteine residue (Cys34) sensitive to oxidation.
• A novel gel electrophoresis-based method (mPEG) has been developed to measure the oxidation state of Cys34.
• The mPEG assay can be run on a drop of blood collected by fingerprick.