Figure 1. Metabolic test.

BLOOD, blood sample; M. BIOPSY, vastus lateralis muscle biopsy. The metabolic study was scheduled to apply and validate the novel one-sample and double-tracer approach (see Methods) for glutathione kinetics assessment in a single muscle biopsy. A [²H₂]glycine primed (26.5 μmol kg⁻¹) infusion (26.5 μmol (kg h)⁻¹) was started at the beginning of the study and a second primed (26.5 μmol kg⁻¹) infusion (26.5 μmol kg⁻¹ h⁻¹) of [¹⁵N]glycine was started 4 h later. Enrichments of precursors ([²H₂]glycine and [¹⁵N]glycine) and of products (l-[¹⁵N]glutathione and l-[²H₂] glutathione) were measured in the single final biopsy (7th hour). l-[¹⁵N]Glutathione enrichment reflects short term tracer incorporation (3 h) while l-[²H₂]glutathione enrichment reflects long term incorporation (7 h). We then calculated muscle glutathione kinetics applying the one-sample, double-tracer equation based on the difference between measured product enrichments (changes over time). For validation of the approach, isotopic enrichments of [²H₂]glycine and l-[²H₂]glutathione were measured in two different blood samples taken 3 and 7 h after the metabolic study began. The traditional equation was applied to calculate glutathione synthesis rate in red blood cells. Blood samples were drawn at baseline, day 7 and day 33 while muscle biopsies were taken at baseline and day 33.