Assessment of Immune and Proteomic Markers in Exhaled Breath Condensate: A Feasibility Study

Exhaled breath condensate (EBC) is a promising matrix for biomarker characterization that reflects the condition of the respiratory system. We assessed the practical feasibility of marker characterization in EBC, including quantification, comparison with plasma and saliva levels, and repeated sampling after six months, focusing on immune markers and proteomics. We measured several immune markers twice, six months apart, in EBC, saliva, and plasma, including cytokines and matrix metalloproteinases, and untargeted proteomics in EBC. α-Amylase activity was almost 10,000-fold lower in EBC than in saliva, suggesting at most limited saliva contamination during EBC collection. Generally, chemokine and eicosanoid levels were lower and more often below the limit of quantification in EBC versus plasma and saliva. Participants were healthy; still, EBC markers could be quantified in ≥50% of samples, except interleukin 12p70 and thromboxane B₂. Correlations across matrices were often low, supporting EBC's matrix-specific properties. EBC proteomic analysis identified up to 209 proteins, several related to lung function. This study highlights EBC as a promising non-invasive method for respiratory biomarker discovery and demonstrates the practical feasibility of detecting immune and proteomic markers in EBC. Additionally, we showed that the EBC proteome can be generated on single EBC samples.