Determination of six thyroid hormones in dog brain and liver using acidic extraction, mixed-mode cleanup, and liquid chromatography?tandem mass spectrometry
Determination of six thyroid hormones in dog brain and liver using acidic extraction, mixed-mode cleanup, and liquid chromatography?tandem mass spectrometry
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采用酸萃取、混合模式净化和液相色谱-串联质谱法测定狗脑和肝脏中的六种甲状腺激素
DOI：
10.1016/j.chroma.2021.462686
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发表时间：
2022
影响因子：
4.1
通讯作者：
Nomiyama Kei
中科院分区：
文献类型：
--
作者：
Takaguchi Kohki;Ono Sumika;Tanoue Rumi;Kunisue Tatsuya;Tanabe Shinsuke;Nomiyama Kei
甲状腺激素（Thyroid hormone，TH）在多种动物的生长、代谢和发育等生理过程中起着重要的调节作用。甲状腺激素原L-甲状腺素（T4）从甲状腺分泌并携带到外周组织。然后T4被生物转化为几种发挥不同作用的代谢产物，主要是通过碘甲状腺原氨酸脱碘酶。测定肝脏和脑等重要器官中的脱碘TH代谢产物有助于了解组织特异性TH代谢和稳态。因此，本研究建立了一种高灵敏度的方法，用于测定储存的狗脑和肝脏中的6种甲状腺激素[T4，3，5，3 ′-三碘-L-甲状腺原氨酸（T3），3，3 ′，5 ′-三碘-L-甲状腺原氨酸（rT 3），3，5-二碘-L-甲状腺原氨酸（3，5-T2），3，3 ′-二碘-L-甲状腺原氨酸（3，3 ′-T2）和3-碘-L-甲状腺原氨酸（3-T1）]。分析方法包括在用甲酸酸化的丙酮中超声辅助提取，用EVOLUTE® EXPRESS CX柱（反相结合强阳离子交换柱）净化，并用液相色谱-串联质谱法定量。脑和肝样品均获得了可接受的准确度（内标校正回收率：80%-120%）以及日内和日间精密度（变异系数：分别<6%和<15%）（n= 3/批次，3天）。此外，脑（0.013-0.12 ng g-1）和肝脏（0.030-0.78 ng g-1）的方法检测限均较低，因此犬脑和肝脏样品中不仅可定量T4、T3和rT 3，还可定量3，3 ′-T2。该方法已成功地应用于分析THs在狗（犬狼疮familiaris）的大脑和肝脏中暴露于多氯联苯（PCBs）。结果表明，多氯联苯暴露组rT 3/T4和3，3 ′-T2/T3浓度比值均显著高于对照组，提示多氯联苯暴露增强了内（酪氨酰）环脱碘（5-脱碘）。在本研究中开发的分析方法，使全面评价外周TH代谢的改变，这是由于暴露于环境污染物。
Thyroid hormones (THs) play a critical role in the regulation of biological processes, such as growth, metabolism, and development, in various animal species. Prohormone L-thyroxine (T4) is secreted from the thyroid gland and carried to peripheral tissues. T4 is then biotransformed to several metabolites which play different roles, mainly by iodothyronine deiodinases. Determination of deiodinated TH metabolites in key organs such as liver and brain would help to understand tissue-specific TH metabolism and homeostasis. In this study, we thus developed a highly sensitive method for the determination of six THs [T4, 3,5,3′-triodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2), 3,3′-diiodo-l-thyronine (3,3′-T2), and 3-iodo-l-thyronine (3-T1)] in the brain and liver by using stored dog samples. The analytical method consisted of ultrasonic-assisted extraction in acetone acidified with formic acid, cleanup with a EVOLUTE® EXPRESS CX cartridge (reversed-phase combined with strong cation-exchange cartridge), and quantification with liquid chromatography-tandem mass spectrometry. Acceptable accuracy (internal standard-corrected recovery: 80%–120%) and intra- and inter-day precision (coefficient of variation: <6% and <15%, respectively) (n= 3/ batch, three days) were obtained for both brain and liver samples. In addition, low method detection limits were achieved for both brain (0.013–0.12 ng g–1) and liver (0.030–0.78 ng g–1), which resulted in the quantitation of not only T4, T3, and rT3, but also 3,3′-T2 in both dog brain and liver samples. The developed method was successfully applied to the analysis of THs in the brain and liver of dogs (Canis lupus familiaris) which were exposed to polychlorinated biphenyls (PCBs). As a result, concentration ratios of rT3/T4 and 3,3′-T2/T3 in the PCB-exposed dogs were significantly higher than those in the control groups, suggesting the enhanced inner (tyrosyl)-ring deiodination (5-deiodination) by PCB exposure. The analytical method developed in the present study enables comprehensive evaluation of alterations in peripheral TH metabolism which are caused by exposure to environmental pollutants.