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

Abstract

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 (2)H from orally supplied (2)H(2)O into stable C-H bonds on the tripeptide. The incorporation of (2)H(2)O 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 (2)H(2)O 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 (2)H(2)O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of (2)H(2)O and a statistical basis for estimating N.

Figure 1

Structure of glutathione-N-ethylmaleimide. The hydrogen atoms indicated by arrows are the 10 C-H bonds that may contribute to N.

Figure 2

Water enrichment in rabbit plasma following an intraperitoneal bolus dose of ²H₂O and ²H₂O in drinking water targeted to result in 5% ²H₂O enriched TBW. Mean ± SEM (smaller than symbols).

Figure 3

Time course of GSH labeling in rabbit blood cells observed as M1 of GSNEM, (composite data for 14 animals). Error bars (SEM) were smaller than the symbols. Data are fit to equation for a filling a single pool in metabolic steady state (see Eq. 5).

Figure 4

Estimating N as the model with best fit to data. Theoretical models were constructed for M0 – M3 for each value of N = 4 to 9. The average error for the fit of plateau data to the models was plotted (red diamonds). The average error for N=0, natural abundance fit is plotted as dashed blue line at y axis. Non-integer values of N correspond to 50/50 mixtures as described in the Results.