Endocrine Abstracts

Thyroid hormones (THs) play a critical role in the regulation of growth and development in both animals and humans. They regulate metabolism primarily through actions in the brain, white fat, brown fat, skeletal muscle, liver and pancreas. Therefore, it is not surprising that since a long time scientists have been interested in measuring TH concentration in different tissues and to relate this value with possible dysfunctions. In general, TH levels are measured by immunoassay (IA)-based methods, however several analytical procedures using liquid chromatography–mass spectrometry (LC–MS) and tandem mass spectrometry (LC–MS/MS) have been developed recently for the quantification of thyroid hormones. These new techniques proved to be more accurate than the IA analysis, but most of them employed rat tissue for TH measurement and required more than 200 mg of sample. This quantity can be considered too high especially when preclinical studies are conducted using mice as test subjects, thus an analytical method that reduces the amount of sample is essential. In this study, we developed a procedure for the quantification of six THs; L-thyroxine (T4), 3,3′,5-triiodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3), 3,5-diiodo-L-thyronine (rT2), 3,3′-diiodo-L-thyronine (T2), 3-iodo-L-thyronine (T1) using isotope (¹³C-T4, ¹³C-T3, ¹³C-rT3, ¹³C-T2) dilution liquid chromatography–mass spectrometry. The major difference with previously described methods lies in the utilization of a nano-UPLC system in micro configuration. This approach lead to a reduction of column internal diameter, flow rate, and injected volume. The results of all these improvements brought a decrease in the amount of sample necessary for the analysis. The method was initially tested to quantify the TH level in liver and then it was extended to kidney, heart, and lung. The new procedure allowed us to measure TH concentration using between 21 and 92 mg of tissue sample. With this method we reduced substantially the amount of tissue necessary for analysis, with respect to analogue methods described in literature. We consider this procedure suitable for analysis of small quantity of samples and it opens the way for a more routine testing of mouse tissues by LC–MS technique.