Figure 3. The formation and fate of succinicGSH.

(a) Schematic representation of GSH biosynthesis in FH-deficient cells on incubation with ¹³C₅-glutamine. The white and black circles under each metabolite illustrate ¹²C- and ¹³C-, respectively. (b) Isotopologue distribution of succinicGSH was measured by LC-MS after incubating cells of the indicated genotype in the presence of ¹³C₅-glutamine for 8 h. (c) GSH and ¹³C₂-fumarate were mixed in a physiological buffer in the presence or absence of protein lysates from FH-deficient cells and the amount of ¹³C₂-succinicGSH was measured by LC-MS. Where indicated, the cell extracts were heat inactivated (h.i.) before the in vitro reaction. (d) The kinetics of GSH and succinicGSH formation from ¹³C₅-Glutamine. (e) The relative level of succinicGSH in the media of mouse cells with the indicated genotype. (f) The kinetics of the formation of the degradation product from GSH was traced by ¹³C₅-Glutamine and assessed by LC-MS. Results were obtained from three independent experiments and represented as average±s.e.m.