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- Title
Measurement of the turnover of glutathione by labeling from 2H-enriched water and LC-MS-MS analysis.
- Authors
Subramanian, Rajan Kombu; Kasumov, Takhar; David, France; Allen Jr., Frederick; Minkler, Paul E.; Hoppel, Charles L.; Anderson, Vernon A.; Brunengraber, Henri
- Abstract
We present a new LC-MS-MS technique for measuring the concentration and stable isotope enrichment of reduced and oxidized glutathione (GSH and GSSG) in biological fluids. Because of differences in signal sensitivity and stability of GSH and GSSG, we assay both compounds as similar GSH thioethers. The assay involves (i) spiking the sample with homoglutathione internal standard (glutamate-cysteine-alanine), (ii) reaction with iodoacetate to form a stable thioether with extant GSH, (iii) reaction with mercaptoethanol to reduce GSSG to GSH, and to trap excess iodoacetate (iv) reaction with excess iodoacetonitrile to form a stable thioether nitrile with GSH derived from GSSG reduction, and (v) electrospray-ionization mass spectrometry of thioethers on a 4000 QTrap (Applied Biosystems) coupled to Agilent HPLC. In addition to enhanced mass spectra, ions monitored are 366.1/237.2 for GSH, 347.2/272.1 for GSH derived from GSSG reduction, and 380.1/251.2 for homoglutathione internal standard (carboxymethyl derivative). The method was applied to the measuring of the turnover of plasma GSH/GSSG in rats whose body fluids were 2% 2H-enriched for 2.5 days. GSH and GSSG showed similar patterns of enrichment with half lives of 21 and 23 hr, respectively.
- Subjects
STABLE isotopes; ISOTOPE separation; GLUTATHIONE; ETHERS; NITRILES; MASS spectrometry
- Publication
FASEB Journal, 2007, Vol 21, Issue 6, pA819
- ISSN
0892-6638
- Publication type
Article