Urine samples from patients were collected at baseline, 2 months, and 6 months of treatment. Urine was stored at −80°C within 1 h of sample collection.65 (link),66 (link) PGE-M and PGD-M are unstable if urine is not stored at −80°C within 90 min of collection. Concentrations of the major urinary prostaglandin (PG)E2 metabolite, PGE-M; the tetranor-PGE1, TN-E, the major urinary metabolite of PGD2, PGD-M; 11-dehydro-thromboxane-B2, dTxB2; the metabolite of PGI2, PGI-M; and leukotriene (LT)E4 were measured in urine at each time point in all study participant. The assays were performed at the Eicosanoid Core Laboratory at Vanderbilt University Medical Center, in the USA. Samples were shipped on dry ice and stored at −80°C until analysis.
[2H6]-PGE-M and [2H11]-TN-E were synthesized as previously described.32 (link) [2H4]-PGI-M and [2H4]-11dTxB2 were purchased from Cayman Chemicals (Ann Arbor, MI USA), and [20,20,20-2H3]-LTE4 from Enzo Life Sciences (Farmingdale, NY USA). Sep-Pak C18 and Oasis HLB (3cc/60mg) extraction cartridges were obtained from Waters Corporation (Milford, MA USA). The organic reagents were of high-performance Liquid Chromatograph (LC) quality and purchased from Sigma Aldrich (St. Louis, MO USA).
[2H6]-PGE-M and [2H11]-TN-E were synthesized as previously described.32 (link) [2H4]-PGI-M and [2H4]-11dTxB2 were purchased from Cayman Chemicals (Ann Arbor, MI USA), and [20,20,20-2H3]-LTE4 from Enzo Life Sciences (Farmingdale, NY USA). Sep-Pak C18 and Oasis HLB (3cc/60mg) extraction cartridges were obtained from Waters Corporation (Milford, MA USA). The organic reagents were of high-performance Liquid Chromatograph (LC) quality and purchased from Sigma Aldrich (St. Louis, MO USA).