(The FASEB Journal. 2008;22:1016.5)
© 2008 FASEB
(The FASEB Journal. 2008;22:1016.5.)
© 2008 FASEB
Measurement of glutathione turnover in human plasma by labeling from 2H-enriched water and LC-MS/MS
RAJAN KOMBU SUBRAMANIAN1,
Rime Abbas3,
France David1,
Juan R. Sanabria4,
Vernon E. Anderson2 and
Henri Brunengraber1
1 Nutrition
2 Biochemistry, Case Westeren Reserve University, Cleveland, OH
3 Surgery, Case Western Reserve University, Cleveland, OH
4 Surgery, University Hospitals CASE Medical Center, Cleveland, OH
ABSTRACT
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 2H incorporation. Using this method, we measured the turnover of plasma GSH/GSSG in human subjects whose body water was 0.5% enriched with 2H 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.
