Elevated airway purines in COPD

Abstract

Background: Adenosine and related purines have established roles in inflammation, and elevated airway concentrations are predicted in patients with COPD. However, accurate airway surface purine measurements can be confounded by stimulation of purine release during collection of typical respiratory samples.

Methods: Airway samples were collected noninvasively as exhaled breath condensate (EBC) from 36 healthy nonsmokers (NS group), 28 healthy smokers (S group), and 89 subjects with COPD (29 with GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage II, 29 with GOLD stage III, and 31 with GOLD stage IV) and analyzed with mass spectrometry for adenosine, adenosine monophosphate (AMP), and phenylalanine, plus urea as a dilution marker. Variable dilution of airway secretions in EBC was controlled using ratios to urea, and airway surface concentrations were calculated using EBC to serum urea-based dilution factors.

Results: EBC adenosine to urea ratios were similar in NS (0.20 ± 0.21) and S (0.22 ± 0.20) groups but elevated in those with COPD (0.32 ± 0.30, P < .01 vs NS). Adenosine to urea ratios were highest in the most severely affected cohort (GOLD IV, 0.35 ± 0.34, P < .01 vs NS) and negatively correlated with FEV(1) (r = -0.27, P < .01). Elevated AMP to urea ratios were also observed in the COPD group (0.58 ± 0.97 COPD, 0.29 ± 0.35 NS, P < .02), but phenylalanine to urea ratios were similar in all groups. Airway surface adenosine concentrations calculated in a subset of subjects were 3.2 ± 2.7 μM in those with COPD (n = 28) relative to 1.7 ± 1.5 μM in the NS group (n = 16, P < .05).

Conclusions: Airway purines are present on airway surfaces at physiologically significant concentrations, are elevated in COPD, and correlate with markers of COPD severity. Purinergic signaling pathways are potential therapeutic targets in COPD, and EBC purines are potential noninvasive biomarkers.

Figure 1.

A, Chromatogram demonstrating detection of urea, adenosine, AMP, and isotopically labeled internal standards within an exhaled breath condensate (EBC) from a subject with COPD. B, Bland-Altman plot of replicate analysis of 17 samples demonstrated that most replicates fell within the 95% limits of agreement (circles and dotted lines represent adenosine to urea ratio; triangles and dashed lines represent AMP to urea ratio). AMP = adenosine monophosphate.

Figure 2.

EBC Ado was successfully measured in 36 healthy NS subjects (NS group), 28 healthy S subjects (S group), and 89 subjects with COPD (29 GOLD II, 29 GOLD III, 31 GOLD IV) and reported as ratios of Ado (nM) to urea (μM) to control for variable dilution of airway secretions. A, Ado to urea ratios differed among the cohorts and were elevated in subjects with COPD relative to NS. B, EBC Phe was measured from EBC from 26 NS group subjects, 29 S group subjects, and 78 subjects with COPD; the Phe to urea ratio (μM Phe to μM urea) was similar among all cohorts. C, Analysis of Ado to urea ratio with subjects with COPD divided by GOLD stage; cohorts differed by analysis of variance (ANOVA) with Ado to urea ratio elevated in GOLD III and GOLD IV subjects relative to NS group subjects. D, A modest correlation (r = −0.27, P = .002) was noted between EBC Ado to urea ratio and FEV₁ % predicted. *P < .05 by ANOVA, †P < .05 vs NS by linear contrast with Tukey-Kramer adjustment. Ado = adenosine; GOLD = Global Initiative for Chronic Obstructive Lung Disease; NS = nonsmoker; Phe = phenylalanine; S = smoker. See Figure 1 legend for expansion of other abbreviation.

Figure 3.

EBC AMP was measured and assessed as ratios of AMP (nM) to urea (μM). A, A trend toward group differences in NS, S, and COPD groups was observed (P = .056 by ANOVA), and EBC AMP to urea ratio was elevated in subjects with COPD vs NS group subjects. B, Analysis of AMP to urea ratio with subjects with COPD stratified by GOLD stage. Cohorts did not differ significantly by ANOVA (P = .18), but a linear trend was noted between EBC AMP to urea ratio and cohort (r = 0.132, P < .05). C, A weak but statistically significant correlation between EBC AMP to urea ratio and percent predicted FEV₁ was noted (r = −0.17, P < .05). †P < .05 vs NS by linear contrast with Tukey-Kramer adjustment. See Figure 1 and 2 legends for expansion of abbreviation.

Figure 4.

A, Concentrations of Ado in airway surface liquid were calculated using EBC to serum urea-based dilution factors. Airway surface Ado concentrations differed among the cohorts and were higher in subjects with COPD relative to NS group subjects. *P < .05 by analysis of variance, ^†P < .05 vs NS. B, Airway Ado concentrations correlated with FEV₁ % predicted (r = −0.39, P < .01). C, EBC urea concentrations (measured after lyophilization) were assessed as a surrogate marker of dilution available in the entire cohort. Values were highly variable but modestly lower in subjects with COPD relative to healthy control subjects. *P < .05 by Mann-Whitney. See Figure 1 and 2 legends for expansion of abbreviations.