Identification of volatile metabolites produced from levodopa metabolism by different bacteria strains of the gut microbiome

Abstract

Interspecies pathways in the gut microbiome have been shown to metabolize levodopa, the primary treatment for Parkinson's disease, and reduce its bioavailability. While the enzymatic reactions have been identified, the ability to establish the resulting macromolecules as biomarkers of microbial metabolism remains technically challenging. In this study, we leveraged an untargeted mass spectrometry-based approach to investigate volatile organic compounds (VOCs) produced during levodopa metabolism by Enterococcus faecalis, Clostridium sporogenes, and Eggerthella lenta. We cultured these organisms with and without their respective bioactive metabolites and detected levodopa-induced shifts in VOC profiles. We then utilized bioinformatics to identify significant differences in 2,6-dimethylpyrazine, 4,6-dimethylpyrimidine, and 4,5-dimethylpyrimidine associated with its biotransformation. Supplementing cultures with inhibitors of levodopa-metabolizing enzymes revealed specific modulation of levodopa-associated diazines, verifying their relationship to its metabolism. Furthermore, functional group analysis depicts strain-specific VOC profiles that reflect interspecies differences in metabolic activity that can be leveraged to assess microbiome functionality in individual patients. Collectively, this work identifies previously uncharacterized metabolites of microbe-mediated levodopa metabolism to determine potential indicators of this activity and further elucidate the metabolic capabilities of different gut bacteria.

Keywords: Gut microbiota; Levodopa; Metabolism; Volatile Organoid Compound.

Fig. 1

VOCs produced during levodopa decarboxylation by E. faecalis: a. Heatmap of volatile metabolites detected from cultures of E. faecalis shows changes in VOC abundance across each experimental condition. b. Principal component analysis shows four distinct clusters of VOC profiles when considering the first two principal components. c, d. Representative chromatograms from each experimental condition and labeled peaks corresponding to statistically significant compounds. e-g. Log transformed abundance of significant VOCs detected from E. faecalis and corresponding FDR-adjusted p-values

Fig. 2

Characterization of VOC profiles generated from C. sporogenes: a. Heatmap of volatile metabolites detected from cultures of C. sporogenes shows changes in VOC abundance across each experimental condition. b. Principal component analysis shows three distinct clusters of VOC profiles when considering the first two principal components, with distinct VOC profiles generated with and without the presence of levodopa. c-f. Representative chromatograms from each experimental condition and labeled peaks corresponding to endogenous VOCs produced from C. sporogenes

Fig. 3

VOCs produced during DHPPA dehydroxylation by E. lenta: a. Heatmap of volatile metabolites detected from cultures of E. lenta shows changes in VOC abundance across each experimental condition. b. Principal component analysis shows four distinct clusters of VOC profiles when considering the first two principal components. c, d. Representative chromatograms from each experimental condition and labeled peaks corresponding to statistically significant compounds. e-g. Log transformed abundance of significant VOCs detected from E. lenta and corresponding FDR-adjusted p-values

Fig. 4

Chemical characterization of strain-specific VOC profiles: a. Heatmap showing differences in VOC abundance across C. sporogenes, E. faecalis, and E. lenta. b. PCA shows three distinct clusters of VOC profiles corresponding to each strain. c. Functional group analyses displays different proportions of VOCs based on the chemical classification