Analysis of volatile compounds in exhaled breath condensate in patients with severe pulmonary arterial hypertension

Abstract

Background: An important challenge to pulmonary arterial hypertension (PAH) diagnosis and treatment is early detection of occult pulmonary vascular pathology. Symptoms are frequently confused with other disease entities that lead to inappropriate interventions and allow for progression to advanced states of disease. There is a significant need to develop new markers for early disease detection and management of PAH.

Methodolgy and findings: Exhaled breath condensate (EBC) samples were compared from 30 age-matched normal healthy individuals and 27 New York Heart Association functional class III and IV idiopathic pulmonary arterial hypertenion (IPAH) patients, a subgroup of PAH. Volatile organic compounds (VOC) in EBC samples were analyzed using gas chromatography/mass spectrometry (GC/MS). Individual peaks in GC profiles were identified in both groups and correlated with pulmonary hemodynamic and clinical endpoints in the IPAH group. Additionally, GC/MS data were analyzed using autoregression followed by partial least squares regression (AR/PLSR) analysis to discriminate between the IPAH and control groups. After correcting for medicaitons, there were 62 unique compounds in the control group, 32 unique compounds in the IPAH group, and 14 in-common compounds between groups. Peak-by-peak analysis of GC profiles of IPAH group EBC samples identified 6 compounds significantly correlated with pulmonary hemodynamic variables important in IPAH diagnosis. AR/PLSR analysis of GC/MS data resulted in a distinct and identifiable metabolic signature for IPAH patients.

Conclusions: These findings indicate the utility of EBC VOC analysis to discriminate between severe IPAH and a healthy population; additionally, we identified potential novel biomarkers that correlated with IPAH pulmonary hemodynamic variables that may be important in screening for less severe forms IPAH.

Figure 1. Representative gas chromatograms from a control subject (black line) and IPAH subject (grey line) (A) showing significantly different (p≤0.05) unique peaks for the control subject (B) and IPAH subject (C).

An example of a head-to-tail comparison of an experimental mass spectrum (D) of one of the identified significantly different peaks unique for the IPAH group at a retention time of 81.436 min. (top) with a NIST/Wiley 2009 database search hit (bottom) identifying N-ethyl-Benzeneamine as giving the best match for the experimental spectrum.

Figure 2. Plot of the results of autoregression and partial least squares analysis-weighted components of control subjects (dark circles) and IPAH subjects (open squares).

22 out of 27 disease (positive) samples were confirmed as positive (sensitivity  = 81.5%) and 21 out of 30 control (negative) samples were confirmed as negative (specificity  = 70.0%). The positive likelihood ratio was 2.76 while the negative likelihood ratio was 0.368.

Figure 3. Plots of step-wise linear regression analysis of actual vs. predicted values for mPAP, PVR, and PAWP.

There were significant associations between mPAP and peak heights at retention times of 90.733 and 96.547 minutes (A); PVR and peak heights at retention times of 90.733, 96.547 and 238.247 minutes (B); PAWP and peak heights at retention times of 67.380 and 118.995 minutes (C).