Department of Radiometry, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Physiol Res. 2014;63(Suppl 1):S133-40. doi: 10.33549/physiolres.932621.
We newly elaborated and adapted several radiometric enzyme assays for the determination of activities of the key enzymes engaged in the biosynthesis (thyroid peroxidase, TPO) and metabolic transformations (conjugating enzymes and iodothyronine deiodinases, IDs) of thyroid hormones (THs) in the thyroid gland and in peripheral tissues, especially in white adipose tissue (WAT). We also elaborated novel, reliable radiometric methods for extremely sensitive determination of enzyme activities of IDs of types 1, 2 and 3 in microsomal fractions of different rat and human tissues, as well as in homogenates of cultured mammalian cells. The use of optimized TLC separation of radioactive products from the unconsumed substrates and film-less autoradiography of radiochromatograms, taking advantage of storage phosphor screens, enabled us to determine IDs enzyme activities as low as 10(-18) katals. In studies of the interaction of fluoxetine (Fluox) with the metabolism of THs, we applied adapted radiometric enzyme assays for iodothyronine sulfotransferases (ST) and uridine 5'-diphospho-glucuronyltransferase (UDP-GT). Fluox is the most frequently used representative of a new group of non-tricyclic antidepressant drugs--selective serotonin re-uptake inhibitors. We used the elaborated assays for quantification the effects of Fluox and for the assessment of the degree of potential induction of rat liver ST and/or UDP-GT enzyme activities by Fluox alone or in combination with T(3). Furthermore, we studied possible changes in IDs activities in murine adipose tissue under the conditions that promoted either tissue hypertrophy (obesogenic treatment) or involution (caloric restriction), and in response to leptin, using our newly developed radiometric enzyme assays for IDs. Our results suggest that deiodinase D1 has a functional role in WAT, with D1 possibly being involved in the control of adipose tissue metabolism and/or accumulation of the tissue. Significant positive correlation between specific enzyme activity of D1 in WAT and plasma leptin levels was found. The newly developed and adapted radiometric enzyme assays proved to be very useful tools for studies of factors modulating THs metabolism, not only in model animals but also in clinical studies of human obesity.
我们新开发并改编了几种放射性酶测定法，用于测定甲状腺中参与甲状腺激素（TH）生物合成（甲状腺过氧化物酶，TPO）和代谢转化（结合酶和碘甲状腺原氨酸脱碘酶，IDs）的关键酶的活性，以及外周组织，特别是白色脂肪组织（WAT）中的活性。我们还开发了新的、可靠的放射性酶法，用于极其灵敏地测定不同大鼠和人组织的微粒体部分以及培养的哺乳动物细胞匀浆中 ID 1、2 和 3 型的酶活性。利用优化的 TLC 分离放射性产物和无膜放射自显影技术，利用存储磷屏，可以将 IDs 酶活性测定低至 10(-18)katals。在研究氟西汀（Fluox）与 TH 代谢相互作用时，我们应用了改编的放射性酶测定法来测定碘甲状腺原氨酸硫转移酶（ST）和尿苷 5'-二磷酸葡糖醛酸基转移酶（UDP-GT）。Fluox 是最常用的新型非三环抗抑郁药——选择性 5-羟色胺再摄取抑制剂的代表之一。我们使用开发的测定法来定量 Fluox 的作用，并评估 Fluox 单独或与 T(3)联合对大鼠肝 ST 和/或 UDP-GT 酶活性的潜在诱导程度。此外，我们使用新开发的 IDs 放射性酶测定法研究了肥胖诱导治疗或热量限制条件下肥胖症促进的脂肪组织肥大或萎缩过程中以及瘦素刺激下，鼠类脂肪组织中 IDs 活性的可能变化。我们的结果表明，脱碘酶 D1 在 WAT 中具有功能作用，D1 可能参与控制脂肪组织代谢和/或组织积累。在 WAT 中 D1 的特定酶活性与血浆瘦素水平之间发现了显著的正相关。新开发和改编的放射性酶测定法被证明是研究调节 THs 代谢的因素的非常有用的工具，不仅在模型动物中，而且在人类肥胖的临床研究中也是如此。
