Oxidative stress or disturbed intracellular redox balance has been linked to several diseases, including Alzheimer's disease, rheumatoid arthritis, acute cerebrovascular disorders, diabetes and cancer. It is characterized by an increase in reactive oxygen species (ROS) such as hydrogen peroxide that can be reduced and disarmed by antioxidants. An antioxidant system is the thioredoxin (Trx) reduction pathway.
Here, we present a microplate-based colorimetric assay that determines the thioredoxin antioxidative capacity. It employs the reduction of Trx which in turn reduces insulin in an NADPH dependent reaction. The insulin sulfhydryl groups react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) resulting in the yellow chromophore TNB. The product correlated with Trx reduction and was measured on a spectrometer-based BMG LABTECH microplate reader.
Using the assay to measure Trx activity in cell lysates of neurons and glial cells revealed a 3.8 fold higher thioredoxin activity in Neurons than compared to glial cells.