Synthesis of GSH-MDI Adducts

Reduced glutathione, GSH (CAS no. 70-18-8) and 4,4′-methylenebis(phenyl isocyanate) or MDI (CAS no. 101-68-8) were from Sigma-Aldrich (St. Louis, MO) and were of ≥98.0% purity. GSH was reacted with MDI under conditions that yield reaction products with the greatest capacity to carbamoylate human albumin, among the conditions tested in previously published studies.^{18 (link)} Briefly, 50 μL of 10% (w/v) MDI in acetone from JT Baker (Phillipsburg, NJ) was added dropwise with stirring to 25 mL of 10 mM GSH in 200 mM sodium phosphate, pH 7.4. The reaction mixture was rotated end-over-end for 2 h at 37 °C and then centrifuged at 10 000g, 0.2 μm filtered, aliquoted, and used immediately or snap frozen in LN₂ and stored at −80 °C until analysis could be performed.