Validation of 8-isoprostane and prostaglandin E₂ measurements in exhaled breath condensate

Abstract

Objective:To qualitatively validate radioimmunoassays for 8-isoprostane and prostaglandin (PG) E₂ in exhaled breath condensate.

Subjects:Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection.

Methods:Samples were pooled together and purified by reverse phase high performance liquid chromatography (RP-HPLC). The eluted fractions were assayed for 8-isoprostane-like immunoreactivity and PGE₂-like immunoreactivity by radioimmunoassays. In addition, simultaneous measurements of exhaled breath condensate unextracted samples with two anti-8-isoprostane and anti-PGE₂ sera with different cross-reactivity were performed.

Results:A single peak of 8-isoprostane-like immunoreactivity and PGE₂-like immunoreactivity co-eluting with 8-isoprostane (retention time: 13 min) and PGE₂ (retention time: 21 min) standards, respectively, was identified by radioimmunoassays. Testing with two different antisera showed similar results for both 8-isoprostane-like immunoreactivity (limits of agreement = 4.5 pg/ml and – 4.1 pg/ml, n = 12) and PGE₂-like immunoreactivity (limits of agreement = 6.1 pg/ ml and – 6.1 pg/ml, n = 12).

Conclusion: This study provides evidence for the specificity of the radioimmunoassays for 8-isoprostane and PGE₂ in exhaled breath condensate. This is critical for proposing these markers as a non-invasive way for monitoring airway inflammation.