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

Abstract

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-[¹⁵N] 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.