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Use of L ‐[ 15 N] glutamic acid and homoglutathione to determine both glutathione synthesis and concentration by gas chromatography‐mass spectrometry (GCMS)
Author(s) -
Humbert Bernard,
Nguyen Patrick,
Obled Christiane,
Bobin Christine,
Vaslin Anne,
Sweeten Shawn,
Darmaun Dominique
Publication year - 2001
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.185
Subject(s) - chemistry , glutathione , chromatography , mass spectrometry , glutamic acid , glutathione disulfide , alanine , gas chromatography–mass spectrometry , quantitative analysis (chemistry) , gas chromatography , amino acid , biochemistry , enzyme
A method for simultaneous measurement of both glutathione enrichment and concentration in a biological sample using gas chromatography mass spectrometry is described. The method is based on the preparation of N,S ‐ethoxycarbonylmethyl ester derivatives of glutathione, and the use of homoglutathione (glutamyl–cysteinyl–alanine) as an internal standard. A procedure for determination of glutamate concentration and enrichment is also reported. Both methods have within‐day and day‐to‐day inter‐assay coefficients of variation less than 5%, and recoveries of known added amounts of glutathione and glutamate are close to 100%. Taken together, these methods allowed determination of glutathione concentration and fractional synthesis rate in red blood cells using L ‐[ 15 N] glutamic acid infusion. This approach was applied in vivo to investigate the effects of a 72 h fast, compared with a control overnight fast, on erythrocyte glutathione in a single dog. The 72 h fast was associated with a 39% decline in erythrocyte glutathione level, (2.9 ± 0.4 versus 4.7 ± 0.5 mmol l −1 , fasting versus control) with no change in glutathione fractional synthesis (67.4 versus 71.3% d −1 , fasting versus control). Copyright © 2001 John Wiley & Sons, Ltd.