COX Inhibition Assay.

The COX assay was performed using a Cayman Chemical COX kit with minor modifications to the recommended protocol. Briefly, COX incubations consisted of 194 μl of reaction buffer, 2 μl of heme, 2 μl of COX (1 or 2) enzyme, 2 μl of AA, and 4 μl of vehicle or increasing concentration of inhibitor. Reactions were conducted for 2 minutes in duplicate at 37°C and were quenched by the addition of 10 μl of 1 M HCl. A 200-μl aliquot of the quenched reaction was transferred into a 1-ml deep-well microplate and diluted with 200 μl of ice-cold methanol. From the diluted mixture, a 100-μl aliquot was mixed with 100 μl of IS in ice-cold methanol, vigorously vortex-mixed, and injected onto the LC–MS/MS. Methodology for detecting analytes of interest were modified from Shinde et al. (2012). The injection volume for all sample types was 10 μl. Rather than merely follow AA depletion, COX inhibition was determined by monitoring the appearance of PGE₂ (PGE₂/IS peak area ratio) in all samples and normalizing the inhibitor response to vehicle controls.