GSH and Cys, as well as their respective oxidized forms, GSSG and Cyss, were determined in the lavage fluid and isolated alveolar macrophages by HPLC analysis. Methods for determination of thiol pairs have been previously described. (Brown et al., 2001;Holguin et al., 1998). In short, 500 μl of BAL fluid from each sample was immediately acidified in cold perchloric acid (5% total), which contained the internal standard γ-glutamyl-glutamate (5 μM; final concentration). The samples were placed on dry ice and then stored at −70°C until ready for analysis by the Brown laboratory. To control for dilution by the lavage procedure, the GSH, GSSG, Cys, and Cyss concentrations in extracellular lung fluid were normalized via the urea method. Blood collection was necessary for quantification of thiol pairs. Approximately 3-5 ml of blood was collected by cardiac puncture from each animal and centrifuged at 10,000 rpm (9300 xg) to expose the plasma, which was used for internal controls required in GSH/GSSG and Cys/CySS redox determinations.
