Refinement of metabolite detection in cystic fibrosis sputum reveals heme negatively correlates with lung function

Summary Pseudomonas aeruginosa lung infections are a leading cause of morbidity and mortality in cystic fibrosis (CF) patients (1, 2). Our laboratory has studied a class of small molecules produced by P. aeruginosa known as phenazines, including pyocyanin and its biogenic precursor phenazine-1-carboxylic acid (PCA). As phenazines are known virulence factors (3), we and others have explored the possibility of using phenazine concentrations as a marker for disease progression (4–6). Previously, we reported that sputum concentrations of pyocyanin and PCA negatively correlate with lung function in cystic fibrosis patients (6). Our study used high performance liquid chromatography (HPLC) to quantify phenazines by UV–vis absorbance after extraction from lung sputum. Since our initial study, methods for metabolite analysis have advanced considerably, aided in large part by usage of mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). Because a more recent study employing LC-MS/MS revealed a surprising decoupling of P. aeruginosa metabolites in sputum and the detection of P. aeruginosa through culturing or microbiome profiles (4), we decided to check whether we could reproduce our previous findings by analyzing sputum samples from a different patient cohort with a new LC-MS instrument in our laboratory. Our new samples were provided by the Mountain West CF Consortium Sputum Biomarker study (7). In the course of performing our new analyses, comparison of our old HPLC data to our new LC-MS data led us to realize that the peak previously assigned to PCA instead originates from heme, and the peak assigned to pyocyanin originates from an as-yet unknown compound. This correction only affects the measurements of phenazines in sputum, and we are confident in the phenazine measurements from isolated cultures and the 16S rRNA gene sequencing data from that study (6). Here we outline the basis for our correction and present additional data showing that heme concentration negatively correlates with lung function in cystic fibrosis patients.