Exploratory Application of Neuropharmacometabolomics in Severe Childhood Traumatic Brain Injury

Abstract

Objectives: To employ metabolomics-based pathway and network analyses to evaluate the cerebrospinal fluid metabolome after severe traumatic brain injury in children and the capacity of combination therapy with probenecid and N-acetylcysteine to impact glutathione-related and other pathways and networks, relative to placebo treatment.

Design: Analysis of cerebrospinal fluid obtained from children enrolled in an Institutional Review Board-approved, randomized, placebo-controlled trial of a combination of probenecid and N-acetylcysteine after severe traumatic brain injury (Trial Registration NCT01322009).

Setting: Thirty-six-bed PICU in a university-affiliated children's hospital.

Patients and subjects: Twelve children 2-18 years old after severe traumatic brain injury and five age-matched control subjects.

Intervention: Probenecid (25 mg/kg) and N-acetylcysteine (140 mg/kg) or placebo administered via naso/orogastric tube.

Measurements and main results: The cerebrospinal fluid metabolome was analyzed in samples from traumatic brain injury patients 24 hours after the first dose of drugs or placebo and control subjects. Feature detection, retention time, alignment, annotation, and principal component analysis and statistical analysis were conducted using XCMS-online. The software "mummichog" was used for pathway and network analyses. A two-component principal component analysis revealed clustering of each of the groups, with distinct metabolomics signatures. Several novel pathways with plausible mechanistic involvement in traumatic brain injury were identified. A combination of metabolomics and pathway/network analyses showed that seven glutathione-centered pathways and two networks were enriched in the cerebrospinal fluid of traumatic brain injury patients treated with probenecid and N-acetylcysteine versus placebo-treated patients. Several additional pathways/networks consisting of components that are known substrates of probenecid-inhibitable transporters were also identified, providing additional mechanistic validation.

Conclusions: This proof-of-concept neuropharmacometabolomics assessment reveals alterations in known and previously unidentified metabolic pathways and supports therapeutic target engagement of the combination of probenecid and N-acetylcysteine treatment after severe traumatic brain injury in children.

Figure 1

PCA analysis based on the first two components shows clustering of the three study groups based on their difference in metabolic profile (blue = control subjects, red = TBI treated with placebo, green = TBI treated with probenecid+NAC). The diameter of each circle represents the DModX value, which indicates the observed distance of a particular individual to the principal components model. XCMS-online metabolomics platform (Scripps Research Institute, San Diego, CA)

Figure 2

Cloud plots of features increased (green) or decreased (red) in placebo treated TBI patients vs. control subjects in positive MS mode (A) and negative MS mode (B). Cloud plots of features increased (green) or decreased (red) in TBI patients treated a combination of probenecid and NAC vs. placebo in positive MS mode (C) and negative MS mode (D). The size of the circle corresponds to the fold-change of the particular feature vs. the reference group and the intensity of the color corresponds inversely to the p-value, with more intense shades representing a smaller p-value. XCMS-online metabolomics platform (Scripps Research Institute, San Diego, CA).

Figure 3

Cloud plot of select pathways affected by TBI vs. control subjects, identified by metabolomics analysis of CSF. The y-axis represents significance (p-value) and the x-axis the percentage of overlapping metabolites. The radius of each circle increases with the number of metabolites relative to the number of metabolites represented by other circles. Excel 2013 (Microsoft Corporation, WA) was used to generate cloud plots of the impacted pathways.

Figure 4

Cloud plot of select pathways affected by treatment with probenecid and NAC vs. placebo in TBI patients, identified by metabolomics analysis of CSF. The y-axis represents significance (p-value) and the x-axis the percentage of overlapping metabolites. The radius of each circle increases with the number of metabolites relative to the number of metabolites represented by other circles. Excel 2013 (Microsoft Corporation, WA) was used to generate cloud plots of the impacted pathways.

Figure 5

Network connectivity of representative modules (1/2 and 1/3) in negative MS mode affected by treatment with probenecid and NAC vs. placebo in TBI patients, identified by metabolomics analysis of CSF. Upregulation of glutathione and a number of its conjugates and connectivity between NADP-dependent pathways are highlighted. Red nodes are upregulated while the blue nodes are downregulated relative to other nodes within individual mode. The color intensity indicates the extent of upregulation or downregulation. Output data from the network analysis were imported to Cytoscape 3.4.0 (18) to generate network visualization.