Elucidation of γ-glutamyl-β-cyanoalanylglycine biosynthesis in mammalian cells by LC-QTOF-MS

γ-Glutamyl-β-cyanoalanylglycine (gEcnAG) is a glutathione analog in which the cysteine moiety in glutathione is replaced with β-cyanoalanine, a known plant cyanide metabolite. Previously, gEcnAG was detected in the liver of rats and chicks exposed to β-cyanoalanine. We reported the detection of gEcnAG in naïve mammalian cells using liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). LC-QTOF-MS analysis enabled high-resolution confirmation (exact mass determination and MS/MS fragmentation) of the gEcnAG structure. The detection of gEcnAG in rat pheochromocytoma (PC12) cells that were not exposed to β-cyanoalanine suggests its endogenous production. Furthermore, the inhibition of myeloperoxidase, an enzyme potentially required for endogenous cyanide generation, decreased gEcnAG levels in PC12 cells. This supports the notion that PC12 cells intrinsically produce cyanide, unlike HepG2 cells, which exhibited lower intracellular gEcnAG levels. Notably, β-cyanoalanine was undetectable in PC12 cells. Moreover, depleting glutathione with buthionine sulfoximine reduced intracellular gEcnAG levels, whereas supplementation with glutathione reduced ethyl ester increased them. These observations suggest that endogenous gEcnAG may be generated from glutathione, potentially through its reaction with endogenous cyanide. Our findings implicate gEcnAG as a possible metabolite of endogenous cyanide.