Advances in the study of biomarkers in canine hypothyroidism

Abstract

Canine hypothyroidism is the most common endocrine disorder in dogs, its characterized by an impaired thyroid hormone synthesis. This disease is often diagnosed through the measurement of thyroid hormones in serum, however, results can be confusing, as thyroid hormones tend to fluctuate. The use of biomarkers leads to new opportunities to achieve a clear diagnosis of diseases, while also providing knowledge on their physiopathology and providing new methods for the monitorization of treatments. This PhD thesis seeks to contribute to the advances in the study of biomarkers in canine hypothyroidism, deepening our knowledge on biomarkers, the methods used for their study and their possible practical applications. The specific objectives are the following • Objective 1: To perform a systematic review on the existing published articles related with biomarkers identified through the use of proteomic analyses in dogs. • Objective 2: To perform a proteomic analysis using serum and saliva from dogs with hypothyroidism and healthy controls, in order to identify possible biomarkers of canine hypothyroidism. • Objective 3: To perform an untargeted metabolomic analysis using serum from dogs with hypothyroidism and healthy controls, and a targeted metabolomic analysis using saliva from dogs with hypothyroidism and healthy controls, in order to identify possible biomarkers of canine hypothyroidism. • Objective 4: To validate the determination of antioxidants and oxidants in two novel sample types, namely; whole blood and red blood cells lysates. • Objective 5: To evaluate the redox status in serum, saliva, whole blood and red blood cells lysates of dogs with hypothyroidism, comparing it with the redox states of non-thyroid diseased dogs and healthy controls. To this end, the methods that were used were: • Saliva samples were collected through the use of polypropylene sponges, meanwhile, serum, whole blood and red blood cells lysates were obtained through yugular venipuncture. • Proteomic analyses were performed in serum and saliva of dogs using liquid proteomics and mass spectrometry. • The untargeted metabolomic analysis was performed in serum and the targeted analysis was performed in saliva, through the use of kits, liquid chromatography and mass spectrometry. • Biomarkers of the redox state were evaluated through spectrophotometric methods using an automatized analyzer and a plate reader. The main results and specific conclusions of this PhD thesis are the following: 1. In a review performed on the published proteomic studies performed in dogs, it was found that, for these, several sample types were used to evaluate the proteome of this specie, more specifically serum and saliva. The use of proteomic analysis to evaluate diseases allows the detection of new biomarkers, as well as, the discovery of new physiopathological routes in the field of endocrine disorders. 2. Hypothyroidism produces changes in the salivary and serum proteome of dogs. Changes in saliva reflect a descent in the metabolic rate, the presence of oxidative stress and of immunological and dermatological alterations, while, changes in serum reflect a reduction in protein synthesis, coagulation, and the presence of hypercholesterolemia. In particular, the activity of LDH, which can be measured easily through a spectrophotometric assay, was found to be decreased in the saliva of hypothyroid dogs in comparison to dogs with other diseases and healthy dogs and could serve as a potential biomarker of canine hypothyroidism. 3. Hypothyroidism produces changes in the salivary and serum metabolome in dogs. Changes in saliva are directly associated with the synthesis of thyroid hormones and an alteration in lipid metabolism, some of the identified metabolites, like tyrosine and phenylalanine could serve as potential biomarkers of canine hypothyroidism. The changes in serum are associated with the pentose phosphate pathway, the biosynthesis of aminoacyl-tARN and pyrimidine metabolism. Some of the identified metabolites, like D-gluconic acid and L-isoleucine could serve as potential biomarkers of canine hypothyroidism. 4. Antioxidants and oxidants can be measured in whole blood and red blood cells lysates of dogs. Dogs with hypothyroidism showed a decrease in CUPRAC, TEAC and thiol, and an increase in AOPP and TBARS in whole blood. They also showed a decrease in TEAC and d-ROMs, and an increase in PON-1 in red blood cells lysates. Additionally, hypothyroidism produces changes in the salivary redox state, with a decreased FRAS and increased TBARS. These results indicate the canine hypothyroidism can produce changes in the redox state and that it can be detected using different sample types.