Use of L-[(15)N] 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 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).
Humbert
B
B
INSERM U. 539, Centre de Recherche en Nutrition Humaine, Nantes, France.
Nguyen
P
P
Obled
C
C
Bobin
C
C
Vaslin
A
A
Sweeten
S
S
Darmaun
D
D
D000818:Animals; D004285:Dogs; D004912:Erythrocytes / Q000737:chemistry; D005215:Fasting; D008401:Gas Chromatography-Mass Spectrometry; D018698:Glutamic Acid / Q000097:blood*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000097:blood*; D006801:Humans; D007700:Kinetics; D009587:Nitrogen Isotopes; D012680:Sensitivity and Specificity
D009587:Nitrogen Isotopes; C402172:homoglutathione; D018698:Glutamic Acid; D005978:Glutathione