Plasma Metabolomics in Human Pulmonary Tuberculosis Disease: A Pilot Study

We aimed to characterize metabolites during tuberculosis (TB) disease and identify new pathophysiologic pathways involved in infection as well as biomarkers of TB onset, progression and resolution. Such data may inform development of new anti-tuberculosis drugs. Plasma samples from adults with newly diagnosed pulmonary TB disease and their matched, asymptomatic, sputum culture-negative household contacts were analyzed using liquid chromatography high-resolution mass spectrometry (LC-MS) to identify metabolites. Statistical and bioinformatics methods were used to select accurate mass/charge (m/z) ions that were significantly different between the two groups at a false discovery rate (FDR) of q<0.05. Two-way hierarchical cluster analysis (HCA) was used to identify clusters of ions contributing to separation of cases and controls, and metabolomics databases were used to match these ions to known metabolites. Identity of specific D-series resolvins, glutamate and Mycobacterium tuberculosis (Mtb)-derived trehalose-6-mycolate was confirmed using LC-MS/MS analysis. Over 23,000 metabolites were detected in untargeted metabolomic analysis and 61 metabolites were significantly different between the two groups. HCA revealed 8 metabolite clusters containing metabolites largely upregulated in patients with TB disease, including anti-TB drugs, glutamate, choline derivatives, Mycobacterium tuberculosis-derived cell wall glycolipids (trehalose-6-mycolate and phosphatidylinositol) and pro-resolving lipid mediators of inflammation, known to stimulate resolution, efferocytosis and microbial killing. The resolvins were confirmed to be RvD1, aspirin-triggered RvD1, and RvD2. This study shows that high-resolution metabolomic analysis can differentiate patients with active TB disease from their asymptomatic household contacts. Specific metabolites upregulated in the plasma of patients with active TB disease, including Mtb-derived glycolipids and resolvins, have potential as biomarkers and may reveal pathways involved in TB disease pathogenesis and resolution

MS/MS fragmentation spectra show positive identification of resolvin D1 (RvD1), resolvin D2 (RvD2), and aspirin-triggered resolvin D1 (AT-RvD1) in plasma from subjects with TB disease.

<p>Metabololipidomics analytical methods that incorporated high-resolution liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS, ABI 5500, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108854#s2" target="_blank">methods</a>) were used to verify these DHA-derived specialized pro-resolving lipid mediators <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108854#pone.0108854-Dalli1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108854#pone.0108854-Yang1" target="_blank">[33]</a>.</p

Linkage of trehalose-6-mycolate, phosphatidylinositol and the D-series resolvin metabolite(s) in plasma of patients without or with MDR-TB.

<p>Linkage of trehalose-6-mycolate, phosphatidylinositol and the D-series resolvin metabolite(s) in plasma of patients without or with MDR-TB.</p

Significant metabolites that distinguish TB patients from household contacts.

<p>(A) Two-way hierarchical cluster analysis (HCA) using C18 chromatography shows 8 clusters of metabolites from human plasma and illustrates the patterns distinguishing those with active TB from household contacts without evidence of TB disease. The 17 subjects with TB disease (TB; shown in green) and the 17 household contacts (HC; shown in red) are shown along the x-axis. (B) Pie chart depicts chemical classes of the 61 significant metabolites from panel 2A according to high-resolution matches to metabolite databases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108854#pone.0108854-Jones1" target="_blank">[6]</a>.</p

Demographic characteristics of individuals with TB disease and their asymptomatic household contacts.

<p>Demographic characteristics of individuals with TB disease and their asymptomatic household contacts.</p