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.