13C isotopic enrichment of glutathione in cell extracts determined by nuclear magnetic resonance spectroscopy

An NMR method was developed for measuring the isotopic enrichment of glutathione in extracts of cells fed a medium containing [3,3'-C-13(2)]cystine. Two sublines of human mammary adenocarcinoma MCF-7 cells were exposed to growth medium containing the labeled cystine for varying periods, treated with monobromobimane, harvested, and extracted with perchloric acid. The glutathione-bimane adduct was partially purified by solid-phase extraction before analysis by H-1 NMR spectroscopy. The isotopic enrichment of the beta-carbon of the cysteinyl residue of glutathione was determined directly in the cell extracts without further purification. These isotopic enrichment data can be used to determine the rate of synthesis of glutathione in cell and tissue extracts. (C) 1999 Academic Press.