Lte4 d5 for lte4

Inflammatory lipid metabolites, such as leukotriene E4 (LTE4), 15-hydroxyeicosatetraenoic acid (15-HETE), thromboxane B2 (TXB2), and prostaglandin F2α (PGF2α) were measured by the liquid chromatography-tandem mass spectrometry method. Particularly, sera collected from asthmatic patients and HCs were separated using a BD Vacutainer^® SST tube (BD Biosciences). LTE4-d5 for LTE4, 15(S)-HETE-d8 for 15-HETE, 11-dehydro TXB2-d4 for 11-dehydro TXB2, and 8-iso PGF2α-d4 for PGF2α (Cayman Chemical Company, Ann Arbor, MI, USA) were used as the internal standards. A Waters Acquity UPLC system (Waters) with a Hypersil GOLD column (ThermoFisher Scientific) was used to separate chromatography. Data were obtained using an API5500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) equipped with an ESI source. Data analysis was performed using Analyst 1.5.2 software (AB Sciex).

Serum and urine samples stored at −70°C were thawed and analyzed by LC-MS/MS. Chromatographic separations of metabolites in urine and serum were performed with a Hypersil GOLD (2.1 X 100 mm, 1.9 μm: ThermoScientific, San Jose, CA, USA). The eluent was introduced into an AB Sciex, API5500 Triple Quadrupole mass spectrometer. The following deuterated internal standards were used: LTE₄-d5 for LTE₄, 15(S)-HETE-d8 for 15-HETE, 11-dehydro TXB₂-d4 for 11-dehydro TXB₂ and 8-iso PGF₂α-d4 for PGF_2α (Cayman Chemical Company, Ann Arbor, MI, USA). In urine samples, creatinine was also quantified to normalize to the actual concentrations of each biomarker and 1,3-dimethyl-2-imidazolidinone (Sigma-Aldrich, St. Louis, MO, USA) and then used as the internal standard for creatinine.