Analysis of Volatile Biomarkers of Airway Inflammation in Breath

Abstract

Breath analysis is non-invasive and acceptable to patients, and is an attractive method for the diagnosis and monitoring of airway inflammation in asthma and COPD. The measurement of the fraction of nitric oxide in exhaled breath (FENO) already has clinical applications because of its association with eosinophilic airway inflammation and the clinical response to corticosteroid, but its role has not been defined in COPD. There may also be other volatile biomarkers of airway inflammation in breath, such as hydrogen sulphide (H2S) and hydrogen cyanide (HCN). These compounds can be analysed in breath using selected ion flow tube–mass spectrometry (SIFT-MS). A study was performed to establish whether FENO levels could predict the clinical response to oral corticosteroid in COPD. A double-blind, crossover ―trial of steroid‖ was undertaken in 65 randomised patients with COPD. The predictive values of FENO for clinically significant changes in six-minute walking distance (6MWD), spirometry (FEV1), and St. George's Respiratory Questionnaire (SGRQ) were calculated. Receiver operator characteristic analysis showed the area under the curve for an increase of 0.2 litres in FEV1 was 0.69 (p=0.04) with an optimum FENO cut point of 50ppb. FENO was not a significant predictor for changes in 6MWD or SGRQ. Experiments were performed to characterise the accuracy, repeatability and dynamic response of the SIFT-MS instrument using acetone as a model volatile compound. Similar experiments were then performed using H2S and HCN. Using a SIFT-MS instrument synchronised with a pneumotachometer, the effects of expiratory flow and volume, and oral vs. nasal passage, on the concentration of a volatile compound in breath were investigated. Using known in vitro acetone concentrations of 600-3000 ppb, there was an instrument measurement bias of 8%, inter-day and intra-day CVs were 5.6% and 0.0% respectively, and the 10-90% response time was 500±50 ms (mean±SE). In 12 healthy volunteers, acetone concentrations at expiratory flows of 193±18 (mean±SD) and 313±32 ml/s were 619±1.83 (geometric mean ± logSD) and 618±1.82 ppb in the fraction 70-85% by volume of exhaled vital capacity (V70-85%), and 636±1.82 and 631±1.83 ppb in V85-100%. For H2S, the mean percentage deviation of SIFT-MS measurements from known concentrations was -12 to -13%. Inter-day and intra-day CVs were 13-22% and 15-25% respectively, and the 10-90% response time was 500±60 ms (mean±SE). For HCN, the mean percentage deviations of SIFT-MS measurements from the known concentrations were -3% to +11%. Inter-day and intra-day CVs were 9-12% and 4-6% respectively, and the dynamic response time was 620±50 ms (mean±SE). Higher concentrations of H2S and HCN were observed in oral vs. nasal exhalations, and the exhaled H2S concentration fell from rapidly after hydrogen peroxide mouthwash. The final experiment compared the concentrations of exhaled H2S and HCN in asthma and COPD patients with control subjects, and determined any relationship between these volatile compounds and biomarkers of airway inflammation. There was no difference in post-mouthwash, nasally-exhaled H2S concentration in six COPD patients vs. six control subjects (2.2±0.4 vs. 2.3±0.3 ppb (mean ± SE)) or in six asthma patients vs. six control subjects (2.1±0.2 vs. 2.2±0.2 ppb). There was no difference in nasally-exhaled HCN concentration in the COPD vs. control groups (3.4±0.3 vs. 3.1±0.4 ppb) or the asthma vs. control groups (4.8±0.4 vs. 4.4±0.8 ppb). In the COPD group, there was a negative correlation between the exhaled H2S concentration and the percentage of neutrophils in sputum (rs=-0.89, p=0.02), while in the control group, a positive correlation between the exhaled H2S concentration and the percentage of neutrophils in sputum approached significance (rs=0.77, p=0.07). The exhaled HCN concentration was negatively correlated with sputum neutrophils in COPD patients (rs=-0.49 to -0.66, p=0.16 to 0.33). Positive correlations were observed between markers of eosinophilic airway inflammation in asthma patients and the concentrations of both H2S (rs=0.6-1.0, p=<0.05 to 0.21) and HCN (rs=0.6-0.8, p=0.16-0.20) in exhaled breath. In conclusion, FENO was a weak predictor of short-term response to oral corticosteroid in COPD, its utility being limited to predicting increase in FEV1. The characteristics of the SIFT-MS analytical technique were appropriate for the on-line analysis of acetone, H2S and HCN, in exhaled breath. On-line SIFT-MS measurement of exhaled acetone concentration required control of expiratory volume but not flow. On-line SIFT-MS measurement of exhaled H2S and HCN concentration required nasal exhalation. While the concentrations of H2S and HCN in exhaled breath did not differ between patient groups and their controls, there were associations between markers of airway inflammation and the concentrations of H2S and HCN in exhaled breath that are worthy of further exploration