Novel method for measurement of glutathione kinetics in neonates using liquid chromatography coupled to isotope ratio mass spectrometry

Authors

  • Henk Schierbeek,

    Corresponding author
    1. Erasmus MC – Sophia Children's Hospital, Department of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
    • Erasmus MC – Sophia Children's Hospital, Department of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
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  • Frans te Braake,

    1. Erasmus MC – Sophia Children's Hospital, Department of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
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  • Jean-Philippe Godin,

    1. Nestlé Research Center, Nestec Ltd., Vers chez les Blanc, Department of Bioanalytical Science, P.O. Box 44, CH-1000 Lausanne 26, Switzerland
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  • Laurent-Bernard Fay,

    1. Nestlé Research Center, Nestec Ltd., Vers chez les Blanc, Department of Bioanalytical Science, P.O. Box 44, CH-1000 Lausanne 26, Switzerland
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  • Johannes B. van Goudoever

    1. Erasmus MC – Sophia Children's Hospital, Department of Neonatology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
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Abstract

A novel analytical method using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) was developed for measuring the fractional synthesis rate (FSR) of glutathione (GSH) in neonates after infusion of [1-13C]-glycine as a tracer. After transformation of GSH into GSSG, its dimeric form, the intra-erythrocytic concentration and 13C-isotopic enrichment of GSH were determined using 200 µL of blood. The results showed that, using LC/IRMS, the concentration (range of µmol/mL) was reliably measured using norvaline as internal standard with precision better than 0.1 µmol/mL. In addition, the 13C-isotopic enrichment measured in the same run gave reliable values with excellent precision (with standard deviation (sd) lower than 0.3‰) and accuracy (measured between 0 and 2 Atom % Excess (APE)). The inter-assay repeatability of δ13C of norvaline used as internal standard with in vivo samples was assessed at −26.07 ± 0.28‰ with coefficient of variance (CV) at 1.1%. The FSR calculated either with GSH or GSSG showed similar results with slightly higher values for GSSG (41.6 ± 4.7 and 46.5 ± 4.4, respectively). The slightly lower FSR of GSH is probably due to interfering compounds in the biological matrix. Successfully used in a clinical study, this rapid and reliable method opens up a variety of kinetic studies with relatively low administration of tracer infusates, reducing the total cost of the study design. The small volume of blood needed enables studies even in extremely small subjects, such as premature infants, as reported in this study. Copyright © 2007 John Wiley & Sons, Ltd.

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