Analysis of glutathione in rat airway surface liquid by capillary zone electrophoresis with conductivity detection

Glutathione (GSH) is an important component of antioxidant defenses in airway surface liquid (ASL), a thin layer (10–30 μm) of liquid covering the epithelial cells lining the airways of the lung. Decreased levels of ASL GSH have been reported in cystic fibrosis (CF), potentially contributing to the severe oxidative stress seen in this disease. To help investigate the role of GSH in ASL, we developed a technique suitable for analysis of GSH and its oxidized form (GSSG) in microliter samples using capillary sampling followed by capillary zone electrophoresis (CZE) analysis with conductivity detection. CZE was carried out in 100 mM CHES and 40 mM lithium hydroxide with 5 mM spermine at pH 9.1 under an applied electric field of −416 V cm−1. To prevent any autooxidation of GSH during sample manipulations, the samples were treated with N-ethylmaleimide (50 mM) to alkylate free thiol (–SH). Under these conditions, GSH and GSSG were cleanly separated without interference from common anions (e.g. Cl−, PO43−, HCO3−, etc.) and the limit of detection for ASL analysis was 11 μM for GSH and 8 μM for GSSG (S/N=3). GSH and GSSG were also measured in rat plasma. Baseline values of 897±210 μM (GSH) and 215±61 μM (GSSG) were obtained for rat ASL (n=8), whereas 12.4±2.7 μM (GSH) and 14.8±6.7 μM (GSSG) were obtained for rat plasma (n=5).