Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Molecular EXposomics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; School of Life Sciences Weihenstephan (Nutrition), Technical University Munich, 85354 Freising, Germany.
Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), Research Unit Comparative Medicine (AVM), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Feb 15;1165:122553. doi: 10.1016/j.jchromb.2021.122553. Epub 2021 Jan 18.
3-iodothyronamine (3-TAM) has been suggested as a novel chemical messenger and potent trace amine-associated receptor 1 ligand in the CNS that occurs naturally as endogenous metabolite of the thyroid hormones. Discrepancies and variations in 3-TAM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-TAM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-TAM and the widely used deuterated quantification standard D-TAM. Here, we report a novel LC-MS/MS method for the quantification of 3-TAM in biological specimens using stable isotope dilution with C-TAM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-TAM. The method detection limit (MDL) and method quantification limit (MQL) of 3-TAM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-TAM were between 85.4% and 94.3%, with a coefficient of variation of 3.7-5.8%. The intra-day and inter-day variations of 3-TAM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-TAM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-TAM values were found when C57BL/6J mice were treated with N-acetyl-3-iodothyronamine or O-acetyl-3-iodothyronamine. Overall, our new stable isotope dilution LC-MS/MS method improves both the sensitivity and selectivity compared with existing methods. The concomitant possibility to quantify additional thyroid hormones such as thyroxine, 3,5,3'-triiodo--thyronine, 3,3',5'-triiodo--thyronine, 3,3'-diiodo--thyronine, and 3,5-diiodo--thyronine further adds to the value of our novel method in exploring the natural occurrence and fate of 3-TAM in biological tissues and fluids.
3-碘甲状腺原氨酸（3-TAM）被认为是一种新型的化学信使和中枢神经系统中的有效微量胺相关受体 1 配体，它天然存在于甲状腺激素的内源性代谢物中。然而，3-TAM 血浆和组织浓度的差异和变化引起了关于 3-TAM 的存在和生物学作用的争议。这些讨论至少部分基于 3-TAM 和广泛使用的氘化定量标准 D-TAM 的不同衰减动力学引起的潜在分析假象。在这里，我们报告了一种使用稳定同位素稀释法（C-TAM）定量生物标本中 3-TAM 的新型 LC-MS/MS 方法，该方法使用新的内标物 C-TAM，其药效动力学特性与内源性 3-TAM 相当。3-TAM 的方法检测限（MDL）和方法定量限（MQL）分别为 0.04 和 0.09 ng/g。3-TAM 的回收率在 85.4%至 94.3%之间，变异系数为 3.7-5.8%。3-TAM 的日内和日间变异分别为 8.45-11.2%和 3.58-5.73%。C57BL/6J 小鼠肝脏内源性 3-TAM 值为 2.20±0.49 pmol/g，检测频率为 50%。当 C57BL/6J 小鼠用 N-乙酰基-3-碘甲状腺原氨酸或 O-乙酰基-3-碘甲状腺原氨酸治疗时，肝脏 3-TAM 值较高。总的来说，与现有方法相比，我们的新稳定同位素稀释 LC-MS/MS 方法提高了灵敏度和选择性。同时能够定量其他甲状腺激素，如甲状腺素、3,5,3'-三碘甲状腺原氨酸、3,3',5'-三碘甲状腺原氨酸、3,3'-二碘甲状腺原氨酸和 3,5-二碘甲状腺原氨酸，进一步增加了我们的新方法在探索 3-TAM 在生物组织和液体中的自然发生和命运中的价值。
