Bronchoalveolar Lavage Fluid from COPD Patients Reveals More Compounds Associated with Disease than Matched Plasma

Abstract

Smoking causes chronic obstructive pulmonary disease (COPD). Though recent studies identified a COPD metabolomic signature in blood, no large studies examine the metabolome in bronchoalveolar lavage (BAL) fluid, a more direct representation of lung cell metabolism. We performed untargeted liquid chromatography-mass spectrometry (LC-MS) on BAL and matched plasma from 115 subjects from the SPIROMICS cohort. Regression was performed with COPD phenotypes as the outcome and metabolites as the predictor, adjusted for clinical covariates and false discovery rate. Weighted gene co-expression network analysis (WGCNA) grouped metabolites into modules which were then associated with phenotypes. K-means clustering grouped similar subjects. We detected 7939 and 10,561 compounds in BAL and paired plasma samples, respectively. FEV₁/FVC (Forced Expiratory Volume in One Second/Forced Vital Capacity) ratio, emphysema, FEV₁ % predicted, and COPD exacerbations associated with 1230, 792, eight, and one BAL compounds, respectively. Only two plasma compounds associated with a COPD phenotype (emphysema). Three BAL co-expression modules associated with FEV₁/FVC and emphysema. K-means BAL metabolomic signature clustering identified two groups, one with more airway obstruction (34% of subjects, median FEV₁/FVC 0.67), one with less (66% of subjects, median FEV₁/FVC 0.77; p < 2 × 10^-4). Associations between metabolites and COPD phenotypes are more robustly represented in BAL compared to plasma.

Keywords: BAL; BALF; COPD; LC–MS; bronchoalveolar lavage; emphysema; mass spectrometry; metabolomics; plasma.

Figure 1

Compilation of Venn diagrams for bronchoalveolar lavage (BAL) and plasma compounds. All compounds (A), only annotated compounds (B), or annotated compounds with identifiers in HMDB (C), or KEGG (D) databases.

Figure 2

BAL and Plasma comparison using distribution of Pearson’s correlation between BAL and plasma. All compounds (left), only annotated compounds (middle), or annotated compounds with KEGG identifiers (right). Mean correlation and t-test p-values are mean = 0.015, P = 1.7 × 10⁻⁴ (A), mean = 0.021, P = 4.7 × 10⁻¹⁵ (B) and mean = 1.1 × 10×, P = 0.94 (C). The set of all compounds passing the <20% missingness preprocessing filter and annotated with HMDB identifiers was equivalent to the corresponding KEGG set, and no separate distribution for HMDB is shown.

Figure 3

Enrichment of compound classes in BAL compounds associated with FEV₁/FVC and % Emphysema). (A) Odds ratio with 95% confidence intervals for compounds in a given category to appear among the FDR corrected FEV₁/FVC associated compounds versus appearing among non-associated compounds, using Fisher’s exact test. Regular expression searches identified compounds of different categories with matching compounds checked manually for accurate categorization. (B) Same as A (top left) but for more specific amino acid containing compounds. Categories shown in B include amino acid containing compounds for amino acids with >10 compounds detected experiment-wide for BAL. In B, a compound need only contain the compound listed to be included. (C) Same as A for % Emphysema (D) Same as B for % Emphysema

Figure 4

Strength of correlation between weighted gene co-expression network analysis (WGCNA) modules and clinical variables and outcomes. Heatmap of module/clinical variable correlation. Plasma (A) or BAL (B). Module colors correspond to dendrogram in Figure S2. Cell text is Pearson correlation (p-value) between the first principal component representing the module and the corresponding variable. Plasma WGCNA modules without any correlation, p-values <0.01, are excluded (12 excluded, 19 displayed) for greater visual clarity. Full plasma WGCNA module to clinical variable correlations are shown in Figure S5.