Simultaneous analysis of 13C-glutathione as its dimeric form GSSG and its precursor [l-13c]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine13C-glutathione as its dimeric form (GSSG) and its precursor [1-13C]glycine in a small volume of erythrocytes in one single analysis. After having transformed13C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the13C-isotopic enrichment of GSSG and glycine in 150 μL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of μmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 μmol/mL. The13C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3%o) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright

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