2474 fluorescence detector

Sample derivatization for HPLC was performed as described previously by Jones [24 (link)] and modified by Harris and Hansen [25 (link)]. Briefly, lung tissue was snap frozen in 5% perchloric acid supplemented with 10 µM gamma-glutamylglutamate. Once thawed, tissue was ultrasonicated then centrifuged at 14000 g for 10 minutes. The supernatant was set aside and the pellet was solubilized in 100 mM sodium hydroxide. Protein concentration was determined by BCA assay. Iodoacetic acid (14.8 mg/mL) was added to the supernatant to block free thiols, the pH was adjusted to 9.0 with saturated potassium tetraborate, and incubated for 20 minutes. Amino groups were labels with dansyl chloride (20 mg/mL) and the lung lysates were incubated at room temperature in the dark overnight. Derivatization was completed by adding 500 µL chloroform followed by vortexing and centrifugation. GSH and GSSG concentrations were resolved and quantified using reverse-phase HPLC (Waters 2695 Alliance Separations Module with a Supelcosil LC-NH₂ column). Detection of peaks was made using a Waters 2474 fluorescence detector (335 nm/518 nm). Molarity values of GSH and GSSG were determined through standardization with the internal standard (gamma-glutamylglutamate) and sample protein concentrations. Redox potentials were calculated using the Nernst equation [26 (link)].

EMB, VYS, YSF, and AF samples were sonicated to homogenize tissues then derivatized following the protocol outlined by Jones (Jones, 2002 (link)) and modified by Harris and Hansen (Harris & Hansen, 2012a (link)). The bicinchoninic acid (BCA) assay using bovine serum albumin as a standard was performed to determine protein content of samples.
GSH, GSSG, Cys and cystine (CySS) concentrations were determined through reverse-phase HPLC analysis using a Waters 2695 Alliance Separations Module (Milford, MA) fitted with a Supelcosil LC-NH₂ column (Sigma-Aldrich; St. Louis, MO). Mobile phase A consisted of 80% methanol (v/v) and 20% ddiH₂O (v/v) and mobile phase B consisted of 62.5% methanol (v/v), 12.5% glacial acetic acid (v/v), and 214mg/ml sodium acetate trihydrate in ddiH₂O. Samples were run at a gradient flow rate of 1ml/min. Detection of peaks was determined using a Waters 2474 fluorescence detector (excitation 335nm and emission at 518nm) with data analysis on Empower 3 software (Waters; Milford, MA).