title: Simultaneous analysis of (13)C-glutathione as its dimeric form GSSG and its precursor [1-(13)C]glycine using liquid chromatography/isotope ratio mass spectrometry.
abstract: 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 determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) 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.
authors: Erasmus Medical Center - Sophia Children's Hospital, Department of Paediatrics, Division of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. h.schierbeek@erasmusmc.nl
authors: Henk
authors: H
authors: Schierbeek
authors: Denise
authors: D
authors: Rook
authors: Frans W J
authors: FW
authors: te Braake
authors: Kristien Y
authors: KY
authors: Dorst
authors: Gardi
authors: G
authors: Voortman
authors: Jean-Philippe
authors: JP
authors: Godin
authors: Laurent-Bernard
authors: LB
authors: Fay
authors: Johannes B
authors: JB
authors: van Goudoever
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D019281:Dimerization; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D007231:Infant, Newborn; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D005978:Glutathione; D005998:Glycine; D019803:Glutathione Disulfide