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Measurement of glutathione turnover in human plasma by labeling from 2H‐enriched water and LC‐MS/MS
Author(s) -
SUBRAMANIAN RAJAN KOMBU,
Abbas Rime,
David France,
Sanabria Juan R.,
Anderson Ver E.,
Brunengraber Henri
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1016.5
Subject(s) - chemistry , glutathione , fragmentation (computing) , derivatization , dithiothreitol , chromatography , selected reaction monitoring , electrospray ionization , mass spectrometry , high performance liquid chromatography , tandem mass spectrometry , biochemistry , enzyme , computer science , operating system
We developed a LC‐MS/MS method for measuring the turnover of reduced and oxidized glutathione (GSH and GSSG) in human plasma. Because of differences in signal sensitivity and stability of GSH and GSSG, we assay both compounds as similar thioethers of GSH. The assay involves derivatization of GSH as carboxymethyl derivative, and GSSG as cyanomethyl derivative after reduction using dithiothreitol. Electrospray‐ionization mass spectrometry of thioethers was performed on a 4000 QTrap (Applied Biosystems) mass spectrometer coupled to an Agilent HPLC. We developed a multiple reaction monitoring (MRM) method based on the M0 and M1 fragmentation pattern. Fragmentation of the M1 peak results in a pair of product ions, i.e. 367.2 → (237.1, 238.1). To determine M1/M0 for the precursor ion, the intensity of both product ions must be summed for the numerator, while the denominator is determined solely by the intensity of the 366.2 → 237.1 fragment ion. The MRM difference of M1/M0 for the minimally labeled GSH and for natural abundance provides a precise quantitation of 2 H incorporation. Using this method, we measured the turnover of plasma GSH/GSSG in human subjects whose body water was 0.5% enriched with 2 H for 72 hr. The half‐lives of labeling of plasma GSH and GSSG, which are mostly of hepatic origin, are 4–5 hr. Supported by NIH grant 1R01ES013925.