2.7. Measurement of total glutathione in lung tissue and epithelial lining fluid

To assess the effects of MWCNT exposure, gender, and Gclm status, we measured levels of the major cellular antioxidant glutathione in lung tissue and ELF. In lung tissue, we measured the total glutathione (GSH+GSSG) content in clarified lung homogenates as previously described [36], using tris-carboxyethyl phosphine to reduce GSSG to GSH, and derivatizing GSH with napthelene-2,3-dicarboxaldehyde. We then measured the relative fluorescence intensity of the derivatized GSH and calculated the GSH levels by interpolating from a standard curve [0.01–0.75 mM]. The total GSH content was normalized to the total protein content of the lung homogenate, which was determined using a commercial Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). The total GSH and protein contents were both determined using triplicate samples of each homogenate.

To determine the concentration of total GSH in ELF, we used essentially the same procedure in BALF as described above for clarified lung homogenates, but with interpolation from a less-concentrated standard curve [0.25–5 μM]. To adjust this concentration for the dilution of ELF during lavage, we calculated the dilution factor by measuring urea in BALF and serum using the QuantiChrom™ Urea Assay Kit (BioAssay Systems, Hayward, CA). The use of urea to calculate a dilution factor assumes that urea freely diffuses between blood and ELF, thereby equalizing the urea concentrations between these compartments [13].