Use of L-[N-15] glutamic acid and homoglutathione to determine both glutathione synthesis and concentration by gas chromatography-mass spectrometry (GCMS)

A method for simultaneous measurement of both glutathione enrichment and co ncentration in a biological sample using gas chromatography mass spectromet ry is described. The method is based on the preparation of N,S-ethoxycarbon ylmethyl ester derivatives of glutathione, and the use of homoglulathione ( glutamyl-cysteinyl-alanine) as an internal standard. A procedure for determ ination of glutamate concentration and enrichment is also reported. Both me thods have within-day and day-to-day interassay coefficients of variation l ess than 5%, and recoveries of known added amounts of glutathione and gluta mate are close to 100%. Taken together, these methods allowed determination of glutathione concentration and fractional synthesis rate in red blood ce lls Using L-[N-15] glutamic acid infusion. This approach was applied in viv o to investigate the effects of a 72 h fast, compared with a control overni ght fast, on erythrocyte glutathione in a single dog. The 72 h fast was ass ociated with a 39% decline in erythrocyte glutathione level, (2.9 +/- 0.4 v ersus 4.7 +/- 0.5 mmol l(-1), fasting versus control) with no change in glu tathione fractional synthesis (67.4 versus 71.3% d(-1), fasting versus cont rol). Copyright (C) 2001 John Wiley & Sons, Ltd.