An Artifact-Free Assay for the GSH/GSSG Ratio Adapted for Finger-Stick Blood Microvolumes: Simple, Sensitive, and Suitable for Any Laboratory

Blood glutathione (GSH), its oxidized form glutathione disulfide (GSSG), and especially the ratio of reduced to oxidized glutathione (GSH/GSSG) are recognized as robust biomarkers of oxidative stress. However, the broader application of these biomarkers has been limited by two major challenges: (1) the high risk of artifact formation during sample handling, which can artificially increase GSSG levels and bias redox balance measurements, and (2) the reliance on complex, instrument-intensive analytical procedures and the requirement for venous blood. We present an adaptation of the highly sensitive and easy-to-perform Tietze recycling method for microvolumes of blood. The challenge is to achieve accurate and precise measurements while avoiding artifacts, taking advantage of the high sensitivity of this enzymatic recycling analytical procedure. The method uses a simplified sample preparation protocol compatible with small blood volumes (up to 10 μL) and requires only basic laboratory equipment, such as a standard spectrophotometer or microplate reader. As this is an enzyme-based assay, we also carefully evaluate the main factors that can affect the measurements. This novel procedure provides a practical tool for monitoring GSH/GSSG as a biomarker of oxidative stress in various experimental settings by eliminating the need for trained personnel for blood sampling (it is suitable for capillary blood), minimizing discomfort for subjects, and avoiding complex procedures or instruments for analyte detection.