Investigation on the role of thyroid hormones and their derivatives in different physiopathogenetic mechanisms using HPLC-MS/MS

High Performance Liquid Chromatography coupled to Tandem Mass Spectrometry (HPLC-MS/MS) is a versatile analytical technique widely applied in chemistry, biology, laboratory medicine, and biochemistry. This integrated system combines liquid chromatography with tandem mass spectrometry, facilitating the effective separation, identification, and quantification of compounds within complex mixtures. The significance of HPLC-MS/MS extends to various fields, including pharmaceuticals, environmental analysis, and metabolites analysis. This research focused on the application of HPLC-MS/MS for the quantification of thyroid hormones (TH), such as 3,5,3’-triiodothyronine (T3) and thyroxine (T4), along with their metabolites. TH play crucial roles in growth processes, development, and energy metabolism, motivating a comprehensive exploration of their dynamics in different physiological and pathological processes. The study is organized around four investigations utilizing HPLC-MS/MS as a pivotal instrument with unique pre-analytical approaches and analytical methods to explore diverse matrices. Firstly, the research unveiled neurocognitive dynamics in adult-onset hypothyroidism, employing a rodent pharmacological model. Using an HPLC-MS/MS method, we quantified the total fraction of T3 and T4 in serum samples, validating the experimental plan and providing insights into the impact of various replacement strategies on neurocognitive and neurobiological alterations. Secondly, the thesis delved into TH dynamics in diabetic retinopathy, utilizing HPLC-MS/MS to detect and quantify T3 and T4 in plasma and retina samples from a type 2 diabetes mouse model. This shed light on potential mechanisms, including T3-dependent microRNA/gene regulatory circuits and mitochondrial function. Thirdly, the study addressed iodine dynamics and the role of enzyme dehalogenase 1 (DEHAL1), employing a Dehal1 KO mouse model. With an adapted method using a derivatization process, our HPLC-MS/MS analysis was able to quantify precursors MIT and DIT in different iodine-supplementation conditions, enhancing understanding of iodine recycling and its consequences. Finally, the thesis investigated DEHAL1 gene mutations in congenital hypothyroidism, using HPLC-MS/MS to measure MIT and DIT levels in serum and urine of patients from different Sudanese families. The findings from the pharmacologically induced hypothyroidism model underscored the necessity and sufficiency of levothyroxine (L-T4) alone to restore memory and neuroprogenitors levels to the euthyroid state, challenging the hypothetical impact on neurogenetic pathways of alternative replacement treatments, such as those implying the use of the metabolite T1AM. Subsequently, we revealed the existence of a local low T3 state (LT3S) in the diabetic retina. This highlighted the TH-dependent response to hyperglycemia-induced stress, unveiling a complex temporal dynamic influencing retinal responses in diabetic retinopathy. Through the development of a new optimized HPLC-MS/MS method, the research uncovered the crucial role of the enzyme DEHAL1 in iodine metabolism within the thyroid gland. Elevated iodotyrosines emerged as biomarkers for the risk of iodine-deficient hypothyroidism, providing translational insights for clinical validation. Eventually, the measurement of serum and urine MIT and DIT emerged as sensitive indicators of iodotyrosines deiodination defects in the presence of DEHAL1/IYD mutations, surpassing traditional urine iodine levels and thyroid function tests. This approach offered valuable diagnostic and management insights into dehalogenase-related disorders, emphasizing the importance of personalized and nuanced approaches. In conclusion, this diverse series of examinations highlighted the instrumental role of HPLC-MS/MS in advancing our understanding of TH dynamics across various physiological and pathological contexts. The precision and versatility of this technique have yielded invaluable insights, paving the way for future research endeavors and potential clinical applications.