HPLC-MS-MS analysis of thyroid hormones and iodotyrosines in knocked-out murine and natural human DEHAL-1 deficiency.

Thyroid hormones (TH), namely 3,5,3’-triiodothyronine (T3) and its precursor thyroxine (T4), are key regulators of growth processes and development, and crucially control the energy metabolism. A proper availability of iodine within the thyroid is crucial for the synthesis of TH in order to maintain the homeostasis of circulating levels. The daily dietary iodine intake is not sufficient to sustain their synthesis and, in fact, most of the intra-thyroidal iodine is recycled through the activity of the Iodotyrosine Dehalogenase-1 (DEHAL-1) enzyme. Mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) precursors produced in excess during TH synthesis, are deiodinated by DEHAL-1, leading to the production of I- and Tyr that are recycled and reused within the thyroid. In the last decade, human mutations of DEHAL1 causing the impairment of its catalytic activity were reported. The clinical picture caused by these mutations recapitulates the phenotype of what is known as Iodotyrosine deiodinase deficiency (ITDD), characterized by hypothyroidism, goitre and, if not treated during childhood, by intellectual impairments. A hallmark of ITDD resides in the increase of urinary and plasmatic MIT and DIT levels. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS) is a powerful technique characterized by high specificity and sensitivity. Even if clinical testing of TH is still performed using immunoassays, various HPLC-MS-MS methods to assay TH at low concentrations have been proposed. HPLC-MS-MS is now emerging as a powerful technique that complements traditional testing overcoming some of their limitations. Hitherto, only one HPLC-MS-MS method have been proposed in order to assay MIT and DIT levels in urine that has been used in the discovery of one of the most recent discovered human DEHAL1 mutations. The aim of my project was to develop a sensitive and robust HPLC-MS-MS method able to quantify MIT and DIT in urine and plasma, together with T3 and T4 in the latter case. The method was extensively validated and used to assay these molecules in urine and plasma collected from the novel Dehal1 knock-out mouse. Our method played a crucial role in the biochemical characterization of this first mammalian model of defective iodine recycling through DEHAL-1 deletion. We detected increased urinary and plasmatic levels of MIT and DIT in the knock-out mice and we demonstrated the presence of elevated concentrations of both molecules even at the first stage of life. The influence of iodine availability was tested, showing that mice were still euthyroid when levels of dietary iodine were sufficiently high. In the presence of scarce iodine availability, coupled to the impaired ability to recycle iodine through DEHAL-1, knock-out mice became hypothyroid. Our results demonstrated the importance of iodine availability in triggering hypothyroidism in the presence of ITDD. The importance of a powerful technique able to detect MIT and DIT was demonstrated assaying these molecules in human urine collected from a consanguineous family with a diagnosed DEHAL-1 deficiency. Our HPLC-MS-MS method was able to detect elevated urinary levels of MIT and DIT in the index patient that was clinically diagnosed with ITDD showing goitre and hypothyroidism. Remarkably, we detected a massive increase of both molecules in urine collected from one of the brothers that was healthy at the time of sample collection but that developed hypothyroidism and goitre several years later. In conclusion, our findings demonstrated the ability of our validated HPLC-MS-MS method to detect MIT, DIT together with T3 and T4 in urine and plasma samples. We showed the potential of MIT and DIT assay, especially in urine, for the early detection of hypothyroidism in DEHAL-1 deficiency. Considering the presence from the beginning of life, the detection of iodotyrosines could be potentially included in the human new-born screening for hypothyroidism.