VOC Profiles of Saliva in Assessment of Halitosis and Submandibular Abscesses Using HS-SPME-GC/MS Technique

Abstract

Halitosis and submandibular abscesses are examples of mouth-related diseases with the possible bacterial origin. Salivary volatile organic compounds (VOCs) are potential biomarkers of them, once they can be addressed as metabolites of bacterial activity. Healthy patients (n = 15), subjects with submandibular abscesses located in fascial deep space (n = 10), and subjects with halitosis (n = 5) were enrolled in the study. Saliva samples were subjected to headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometry (GC/MS) analysis. A total number of 164 VOCs was detected by the developed methodology, 23 specific for halitosis and 41 for abscess. Halitosis' profiles were characterized by a larger number of sulfur compounds, while for abscess they had a higher variety of alcohols, aldehydes, and hydrocarbons-biomarkers of inflammatory processes. Principal components analysis allowed visualization of clusters formed according to the evaluated conditions. Kruskal-Wallis test indicated that 39 VOCs presented differentiated responses between the studied groups, with statistical relevance (p < 0.05). Random forest was applied, and a prediction model based on eight VOCs (2-butanone, methyl thioacetate, 2-methylbutanoic acid, S-methyl pentanethioate, dimethyl tetrasulfide, indolizine, pentadecane, and octadecanal) provided 100% of sensitivity, 82% of specificity, and 91% of balanced accuracy, indicating the specific presence of submandibular abscess.

Keywords: GC/MS; VOCs; diagnosis; halitosis; saliva; submandibular abscess.

Figure 1

Functional group distribution of VOCs (volatile organic compounds) for healthy patients (HE), subjects with submandibular abscesses (AB), and halitosis (HA), where: VNCs—volatile nitrogen compounds, VSCs—volatile sulfur compounds.

Figure 2

Correlation networks of VOCs emitted from saliva samples from all three group of patients, where: healthy patients (HE), subjects with submandibular abscesses (AB) and halitosis (HA). VOCs are shown as nodes. Numbers in circles denote the number of volatiles from each group.

Figure 3

Hierarchical cluster analysis and heatmap of VOC profiles based on peak areas from chromatograms. Columns represent three groups of patients (total of 30 individuals): healthy (15), segregated from an abscess (10) and halitosis (5). Rows represent 39 discriminating VOCs (orange, low abundance; red, high abundance).

Figure 4

PCA (Principal Component Analysis) plot depicting the differentiation of group of individuals by VOC profiles; Assigned clusters: healthy controls (HE), patients with submandibular abscess (AB) and halitosis (HA).

Figure 5

Assessment of variable importance, in terms of (A) mean decrease in accuracy and (B) mean decrease Gini or node impurity.

Figure 6

(A) Exemplary decision tree produced in Random Forest approach; (B) Overlaid ROC (receiver operating characteristic) curves concerning discrimination of specific condition (in green- control / “healthy”, in red- abscess, and in blue- halitosis) against all others.