Non-targeted metabolite profiling reveals changes in oxidative stress, tryptophan and lipid metabolisms in fearful dogs

Abstract

Background: Anxieties, such as shyness, noise phobia and separation anxiety, are common but poorly understood behavioural problems in domestic dogs, Canis familiaris. Although studies have demonstrated genetic and environmental contributions to anxiety pathogenesis, better understanding of the molecular underpinnings is needed to improve diagnostics, management and treatment plans. As a part of our ongoing canine anxiety genetics efforts, this study aimed to pilot a metabolomics approach in fearful and non-fearful dogs to identify candidate biomarkers for more objective phenotyping purposes and to refer to potential underlying biological problem.

Methods: We collected whole blood samples from 10 fearful and 10 non-fearful Great Danes and performed a liquid chromatography combined with mass spectrometry (LC-MS)-based non-targeted metabolite profiling.

Results: Non-targeted metabolomics analysis detected six 932 metabolite entities in four analytical modes [RP and HILIC; ESI(-) and ESI(+)], of which 239 differed statistically between the test groups. We identified changes in 13 metabolites (fold change ranging from 1.28 to 2.85) between fearful and non-fearful dogs, including hypoxanthine, indoxylsulfate and several phospholipids. These molecules are involved in oxidative stress, tryptophan and lipid metabolisms.

Conclusions: We identified significant alterations in the metabolism of fearful dogs, and some of these changes appear relevant to anxiety also in other species. This pilot study demonstrates the feasibility of the non-targeted metabolomics and warrants a larger replication study to confirm the role of the identified biomarkers and pathways in canine anxiety.

Fig. 1

Partial least squares discriminant analysis (PLS-DA) of the reversed phase positive ESI–MS mode data. The score plot shows the individual samples in both case and control groups. Case group (black circles); Control group (grey squares)

Fig. 2

K-means cluster analysis with the hierarchical clustering of the fold change values (case versus control). Included are the metabolic features having p value <0.05 and fold change value ≥±1.2. The clusters are numbered one–four as depicted in the figure. The values for peak areas were row-wise normalized, and the colour scale indicates high (red) or (low) metabolite level. Grey colour in the heat map indicates not detected value