Trapping Reactive Metabolites Using Glutathione

The reactive metabolites were trapped with GSH in our current study. The experiments were conducted in 1× PBS (pH 7.4), containing 30 μM sepin-1, 1.0 mg HLM, and GSH (2.5 mM) in a final volume of 190 μl. After 5 min of pre-incubation at 37°C, the reactions were initiated by the addition of 10 μl of 20 mM NADPH (final concentration 1.0 mM) and continued for 30 min with gentle shaking. Incubations in the absence of NADPH and trapping agents were used as controls. The reactions were quenched by adding 200 μl of ice-cold methanol. The mixtures were vortexed for one 30 s and centrifuged at 15,000 × g for 15 min. The supernatants were transferred to sample vials for analysis. Incubations were performed in triplicate.