Exhaled breath condensate: a promising source for biomarkers of lung disease

Abstract

Exhaled breath condensate (EBC) has been increasingly studied as a noninvasive research method for sampling the alveolar and airway space and is recognized as a promising source of biomarkers of lung diseases. Substances measured in EBC include oxidative stress and inflammatory mediators, such as arachidonic acid derivatives, reactive oxygen/nitrogen species, reduced and oxidized glutathione, and inflammatory cytokines. Although EBC has great potential as a source of biomarkers in many lung diseases, the low concentrations of compounds within the EBC present challenges in sample collection and analysis. Although EBC is viewed as a noninvasive method for sampling airway lining fluid (ALF), validation is necessary to confirm that EBC truly represents the ALF. Likewise, a dilution factor for the EBC is needed in order to compare across subjects and determine changes in the ALF. The aims of this paper are to address the characteristics of EBC; strategies to standardize EBC sample collection and review available analytical techniques for EBC analysis.

Figure 1

Nonvolatile and volatile components in EBC. Water vapor is rapidly diffused from the lining fluid on the surface of the airway (bronchi) and airspace (alveolar) into the expiratory flow. Droplets (nonvolatile biomarker) formation in the lung is largely from the lining fluid of the airway where turbulence is encountered. Respiratory gases (volatile biomarkers) are from both airspace and airway, and more soluble vapors are typically greater in the airway [5, 67].

Figure 2

Metabolism of arachidonic acid. Arachidonic acid is released from phospholipids by the action of phospholipase A2 (PLA2). Arachidonic acid is metabolized by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP). COXs metabolize arachidonic acid to prostaglandins (PGs), prostacyclin (PGI2), and thromboxanes (TXs). Leukotrienes (LTs) are the final arachidonic acid metabolites in the 5-lipoxygenase- (5-LO-) mediated pathway. CYP epoxygenases (CYP-EO) metabolize arachidonic acid to epoxyeicosatrienoic acid (EETs), and CYP hydroxylases (CYP-HO) metabolize arachidonic acid to hydroxyeicosatetraenoic acids (HETEs). 8-Isoprostane (8-IP) can be generated in vivo by the free radical-catalyzed peroxidation of arachidonic acid.

Figure 3

Reactive oxygen and nitrogen species and redox relevant molecules in EBC. Exhaled nitric oxide (NO) is derived from L-arginine by enzyme nitric oxide synthase (NOS). NO can combine with superoxide (•O₂ ⁻) to form peroxynitrite (ONOO⁻). ONOO⁻ induces nitrosation of tyrosine (Tyr) residues and forms 3-nitrotyrosine (3-NT). NO can also react with thiols to form S-nitroso thiols (RS-NO). The end-products of NO are nitrite (NO₂ ⁻) or nitrate (NO₃ ⁻). •O₂ ⁻ is one of major reactive oxygen species generated from NADPH oxidase (NOX) or mitochondrial electron transfer chain. •O₂ ⁻ is converted to hydrogen peroxide (H₂O₂) by superoxide dismutases (SOD). H₂O₂ can be converted to the highly reactive hydroxyl radical (•OH), which is catalyzed by Fe²⁺ (Fenton reaction). H₂O₂ can be removed by thiol-specific antioxidant enzymes to form water.