Discovery of Volatile Biomarkers of Parkinson's Disease from Sebum

Abstract

Parkinson's disease (PD) is a progressive, neurodegenerative disease that presents with significant motor symptoms, for which there is no diagnostic chemical test. We have serendipitously identified a hyperosmic individual, a "Super Smeller" who can detect PD by odor alone, and our early pilot studies have indicated that the odor was present in the sebum from the skin of PD subjects. Here, we have employed an unbiased approach to investigate the volatile metabolites of sebum samples obtained noninvasively from the upper back of 64 participants in total (21 controls and 43 PD subjects). Our results, validated by an independent cohort (n=31), identified a distinct volatiles-associated signature of PD, including altered levels of perillic aldehyde and eicosane, the smell of which was then described as being highly similar to the scent of PD by our "Super Smeller".

Figure 1

Schematic outline of the workflow described in this study - from sample collection to biomarker discovery. Parkinson’s disease patient samples and control participant samples were collected from 25 sites across the UK using gauze swabs to sample the sebum from the top back region from 64 people. Thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) analysis was performed alongside olfactory analysis, results of which were then combined. Statistical analysis was performed on two independent cohorts. Data from discovery cohort consisting of 30 participants were used to create a partial least-squares-discriminant analysis (PLS-DA) model, and differential features found as a result were then targeted for the presence in a separate validation cohort consisting of 31 participants. The significance of these biomarkers was tested using receiver operating characteristic (ROC) analyses and the Wilcoxon-Mann–Whitney test. Finally, four features that showed similar statistical significance and expression in both cohorts were selected for biological interpretation of data.

Figure 2

PLS–DA classification model. (a) Classification matrix of PLS–DA model validated using 5-fold cross validation showing 90% correct prediction of Parkinson’s disease samples. (b) PLS–DA modeling was further tested using permutation tests (where the output classification was randomized; n = 28), and results were plotted as a histogram which showed frequency distribution of correct classification rate (CCR) which yielded CCRs ranging between 0.4 and 0.9 for permutated models. The observed model was significantly better than most of the permuted models (p < 0.1), shown by the red arrow. (c) ROC plot generated using combined samples from both cohorts and the panel of four metabolites that was common and differential between control and PD. The shaded blue area indicates 95% confidence intervals calculated by Monte Carlo cross validation (MCCV) using balanced subsampling with multiple repeats.

Figure 3

ROC curves and box plots for analytes of interest: In each panel from top to bottom: ROC curves for both discovery (blue) and validation (red) cohort for four analytes common to both experiments. Confidence intervals were computed with 2000 stratified bootstrap replicates, and diagonal black line represents random guess. Box plots show comparison of means of log scaled peak intensities of these analytes, where black dots were outliers.

Figure 4

Comparison of GC–MS chromatogram to description of olfactory data described by the Super Smeller: GC–MS chromatogram from three drug naïve Parkinson’s subjects and a blank gauze. (A) The 10–25 min retention time range of the chromatographic analysis in which the Super Smeller described various odors associated with different GC–MS peaks. The overlaid green shaded area shows the overlap between real time GC–MS analysis and the Super Smeller describing a “strong PD smell” via the odor port. (B) A zoom of the green highlighted area from A. This region is of particular interest as three out of four identified compounds are found here (Tables 1 and S2); it encompasses the time during which the Super Smeller described a musky PD-like scent as being “very strong” (between the time lines at 19 and 21 min) for the PD samples and not for the blank. It can be noted that none of these compounds are found in blank gauze (bottom chromatogram) within the same retention time window as shown by normalized relative peak intensities to the highest peak in each chromatogram. The area between black dotted lines highlights the presence of compounds in PD samples but complete absence in the blank gauze.