Assessment of Glutathione Homeostasis

The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the most abundant antioxidant thiol in the brain. GSH plays a critical role in protecting brain cells from oxidative stress and xenobiotics, as well as maintaining the thiol redox state. High levels of GSH are present in the central nervous system, particularly in astrocytes. GSH is synthesized into two enzymatic steps, the first, and the rate-limiting one, is catalyzed by glutamate–cysteine ligase (GCL) to form a dipeptide which is then converted to GSH by GSH synthetase. In this chapter, we describe assays for the measurements of GSH levels and GCL activity. The first spectrophotometric assay is based on the affinity of 2,3-naphthalenedicarboxaldehyde (NDA) for GSH. In the second assay, GSH levels are measured after being derivatized using the fluorescent thiol reactive compound monobromobimane (MBB) and are detected by high-performance liquid chromatography (HPLC). The third assay allows the assessment of GCL activity, also by HPLC.