Measurement of eicosanoid metabolites
Utilizing established solid phase extraction approach for the collection and purification of eicosanoids in EBCs, a panel of 6 eicosanoid species derived from arachidonic acids, representing products from major enzymatic pathways, was selected for initial discovery phase of the study population consisting of 60 asthmatics and 20 healthy controls, who were randomly selected from among the study populations. Each of the selected eicosanoid species was then measured, depending upon the availability of the EBC samples. Eicosanoids were extracted from EBC with C18 Sep-Pak cartridges (Waters, Milford, Massachusetts) and concentrated as previously described15. The levels of eicosanoids were measured with the respective enzyme immunoassay kit (Cayman Chemical, Ann Arbor, Michigan and Neogen, Lexington, Ky) as recommended by the manufacturer. The lower limits of detection for the assays were 3.9 pg/ml for LTB4, 7.8 pg/ml for LTE4, 7.8 pg/ml for PGE2, 20 pg/ml for LXA4, 1.6 pg/ml for TXB2, and 39 pg/ml for 15-HETE. When the level in samples was below the detection limit, the detection limit for the respective measurement was then assigned to each sample.