MSACL 2025 Abstract

Plasma Metabolomic and Proteomic Profiling of Anxious Dogs by HPLC-MS/MS: A Case-Control Study

Claudia Gaither (1,2), Robert Popp (2), Francis Beaudry (1,3), Christoph H. Borchers (4-6), and Marion Desmarchelier (1)
(1) Faculté de médecine vétérinaire – Département des sciences cliniques, Université de Montréal, Canada, (2) MRM Proteomics Inc., Montréal, Canada, (3) Centre de recherche sur le cerveau et l’apprentissage (CIRCA), Université de Montréal, Montréal, Canada, (4) Division of Experimental Medicine, McGill University, Montréal, Canada, (5) Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Canada, (6) Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Canada

Claudia Gaither, MSc, BSc, Dipl. ACBT (Presenter) Faculté de médecine vétérinaire, Université de Montréal >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months, reported on Mar 19, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
The prevalence of anxiety disorders in dogs highlights the need for novel/better diagnostics, prediction of treatment success, and treatment progress monitoring that can be achieved using innovative MS-based analyses. Behavioral problems affect up to 85% of dogs, with many stemming from underlying anxiety disorders. Some of the most common anxiety-related behavioral problems are separation anxiety, generalized anxiety, aggression, and compulsive behaviors. Although MS has been used for some disease profiling experiments in dogs and other species, it is usually overlooked in the veterinary and animal science fields, particularly in the animal behavior and psychiatry disciplines. Thus, here we investigate plasma proteomic and metabolomic differences between a group of anxious dogs and a group of non-anxious dogs.

METHODS:
Venous blood was collected, and plasma generated at the Faculté de médecine vétérinaire, Université de Montréal. Ten control and ten patient dogs underwent behavioral evaluations by a board-certified veterinary behaviorist and either diagnosed with an anxiety disorder or confirmed lack thereof, respectively. Samples were prepared for proteomic and metabolomic analyses by in-solution enzymatic digestion and solvent extraction of metabolites, respectively, for untargeted HPLC-MS/MS. Label-free quantitative MS data were acquired using a Vanquish Flex UHPLC, and an Evosep One system, both interfaced to a Q Exactive Plus Orbitrap MS (Thermo). Data were analyzed using Proteome Discoverer 2.2 or Compound Discoverer 2.1 (Thermo). Plasma protein and metabolite differences between the groups were assessed based on fold changes and t-tests (p-value ≤ 0.05).

RESULTS:
A total of 49 proteins had statistically significantly different plasma levels between anxious and non-anxious/control dogs, as determined by two-tailed student t-tests (p-value ≤ 0.05). Among the differential proteins were Apolipoproteins A-II, C-I and C-IV, involved in the packaging, transporting, and metabolizing of lipids, including cholesterol. Cholesterol is the precursor for cortisol, a well-studied biomarker for stress that leads to various physiological effects, including the flight or fight response. Other proteins found to be at different levels between the two groups were Fibrinogen Beta and Gamma chains, Serine Protease Inhibitors and Coagulation Factor XII, which are part of the blood coagulation cascade. These higher levels could be a result from cortisol production resulting in dysfunctional coagulation and excess clot formation. Finally, Complement Factors I and D, complement components C5, C6, C8A, and Complement Component 4 Binding Protein (C4BP) were differential and are involved in the complement system cascade, part of the innate immune response. The activation of C3 and C5 convertases is regulated by C4BP, and its differential expression could result in dysfunctional regulation of such enzymes, leading to increased or continuous inflammation.

Preliminary metabolomics results also show plasma profile differences between the two groups of dogs. Some of the metabolites that appear to be higher in the anxious dogs include phenylalanine, creatine, certain bile acids and certain fatty acids. In humans, higher levels of phenylalanine, a precursor to catecholamine neurotransmitters, are linked to anxiety. Bile acids can directly bind to brain receptors through the blood-brain barrier and their signaling may induce anxiety. Long chain saturated fatty acids are linked to anxiety-like behavior in mice and other fatty acids promote inflammation. Finally, metabolites like adenosine were lower in the anxious dogs, compared to controls. Low adenosine in humans can decrease sleep quality thus resulting in anxiety and depression.

CONCLUSION:
To our knowledge, this is the first unbiased/comprehensive clinical in-depth metabolomic/proteomic profiling of plasma from dogs with anxiety disorders. On the proteomics side, there are three main pathways that appear differential between the control and anxious dogs: those involved in lipid metabolism, the coagulation cascade, and the complement system. On the metabolomics side, phenylalanine, creatine, certain bile acids and certain fatty acids were found to be differential. Targeted assays will need to be developed for the validation of our findings and bring the field a step closer to elucidating the pathophysiology of anxiety.