Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of ²H from orally supplied ²H₂O into stable C-H bonds on the tripeptide. The incorporation of ²H₂O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with ²H₂O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N = 6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with ²H₂O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of ²H₂O and a statistical basis for estimating N.