Lung function tests, methacholine challenge tests and analysis of eicosanoids
The lung function tests were performed with the spirometer-Lungest 1000 (MES, Krakow, Poland) according to ERS/ATS14standards. These children had not received any anti-asthmatic medication, including oral steroids, for at least two days.
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. Eicosanoids were extracted from EBCs15 and measured with the respective enzyme immunoassay kit (Cayman Chemical, Ann Arbor, Michigan and Neogen, Lexington, Ky) as described previously11.