Plasma metabolomic biomarkers accurately classify acute mild traumatic brain injury from controls

Abstract

Past and recent attempts at devising objective biomarkers for traumatic brain injury (TBI) in both blood and cerebrospinal fluid have focused on abundance measures of time-dependent proteins. Similar independent determinants would be most welcome in diagnosing the most common form of TBI, mild TBI (mTBI), which remains difficult to define and confirm based solely on clinical criteria. There are currently no consensus diagnostic measures that objectively define individuals as having sustained an acute mTBI. Plasma metabolomic analyses have recently evolved to offer an alternative to proteomic analyses, offering an orthogonal diagnostic measure to what is currently available. The purpose of this study was to determine whether a developed set of metabolomic biomarkers is able to objectively classify college athletes sustaining mTBI from non-injured teammates, within 6 hours of trauma and whether such a biomarker panel could be effectively applied to an independent cohort of TBI and control subjects. A 6-metabolite panel was developed from biomarkers that had their identities confirmed using tandem mass spectrometry (MS/MS) in our Athlete cohort. These biomarkers were defined at ≤6 hours following mTBI and objectively classified mTBI athletes from teammate controls, and provided similar classification of these groups at the 2, 3, and 7 days post-mTBI. The same 6-metabolite panel, when applied to a separate, independent cohort provided statistically similar results despite major differences between the two cohorts. Our confirmed plasma biomarker panel objectively classifies acute mTBI cases from controls within 6 hours of injury in our two independent cohorts. While encouraged by our initial results, we expect future studies to expand on these initial observations.

Conflict of interest statement

Competing Interests: MSF, MM, KM-B, JB, and HJF have filed intellectual property related to this research through Georgetown University. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other co-authors have declared no competing interests exist.

Figures

Fig 1. College Athlete cohort–metabolomic biomarker study design.

In the college athlete cohort (Athletes), the mTBI (mild traumatic brain injury) and NC (non-concussed control) groups were definitively identified as a result of longitudinal clinical assessment of study participants throughout their sports seasons. Identification of mTBI during the Season allowed retrospective designation of group participants in the Preseason. Analytic timepoints following mTBI occurrence are indicated as ≤6h = ≤6 hours; 2d = 2 days; 3d = 3 days; and 7d = 7 days.

Fig 2. Athlete cohort preliminary multivariate ROC AUC analysis plots.

Example college athlete cohort (Athletes) receiver operating characteristic (ROC) area under the curve (AUC) analysis results from the MetaboAnalyst 3.0 Explorer function of Biomarker Analysis module, with plots of sensitivity (y-axis) and 1-specificity (x-axis). In A-C, the plots indicate no significant difference between the CAC Preseason mild traumatic brain injury (mTBI) and non-concussed subjects (NCS) groups using either (i) Linear support vector machine (SVM), (ii) partial least squares discriminant analysis (PLS-DA), or (iii) random forests methods. ROC AUC values in all three analyses are ~ 0.5. The legend at lower right of each graph indicates AUC and 95% confidence interval (CI) values for derived models using 5–100 analytes (Var.). Plots D-F provide examples of more significant differences between comparison groups, such as between Athlete Preseason NC (non-concussed teammate controls) and the Season NC groups, using the same analytic methods as A-C, but with ROC AUC results ranging from about 0.70 to 0.86.