Determination of Blood Total, Reduced, and Oxidized Glutathione in Pediatric Subjects

Abstract

Glutathione (l-γ-glutamyl-l-cysteinylglycine), which is present in virtually all mammalian tissues, provides reducing capacity for several reactions and plays an important role in detoxification of hydrogen peroxide, other peroxides, and free radicals (1). The synthesis and degradation of glutathione are controlled by reactions of the γ-glutamyl cycle; a decrease in blood reduced glutathione (GSH) has been reported in patients affected by deficiencies of the enzymes involved in the synthesis of glutathione (1). In cells, total glutathione can be free or bound to proteins; measurement of free glutathione in blood samples is essential for evaluation of the redox and detoxification status of cells in relation to its protective role against oxidative and free radical-mediated cell injury; moreover, GSH measurement is important for the diagnosis of γ-glutamyl cycle disorders. Recently, several methods to measure glutathione in blood have been described, but little is known about the concentrations of various forms of blood glutathione in pediatric subjects (2)(3)(4)(5)(6). We report a rapid and fully automated HPLC method for determining total (tGSH), reduced (GSH), and oxidized glutathione (GSSG) in whole blood. Moreover, we report values for the different forms of blood glutathione in pediatric subjects. The reference sample group included 227 healthy, randomly selected children who attended the outpatient clinic of our hospital. The children were selected to provide approximately equal numbers of each sex across the age range of 0–15 years. The study was performed according to the recommendations of the Ethics Committee of Children’s Hospital and Research Institute “Bambino Gesu”. Blood was collected into a Vacutainer Tube (Becton Dickinson) containing EDTA. The hemoglobin …