Department of Pathology, University of Pisa , Pisa, Italy .
Thyroid. 2017 Oct;27(10):1323-1331. doi: 10.1089/thy.2017.0111.
Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes.
Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters.
In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood.
These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.
在血液或血清中定量检测 3-碘甲状腺原氨酸胺（T1AM）存在困难，据推测这是由于提取或其他分析前步骤的问题所致。出于这个原因，即使是细胞培养实验也常常使用非生理的无蛋白培养基进行。本研究的目的是评估添加到标准细胞培养基（即含有胎牛血清（FBS）的 Dulbecco 最小必需培养基（DMEM））和其他生物基质中的外源性 T1AM 的回收率。
细胞培养基（Krebs-Ringer 缓冲液、DMEM、FBS、DMEM+FBS，无论是在没有 NG108-15 细胞的情况下还是在存在 NG108-15 细胞的情况下使用）和其他生物基质（大鼠脑和肝匀浆、人血浆和血液）中添加 T1AM 和/或氘代 T1AM（d4-T1AM）并孵育 0 至 240 分钟。然后使用液/液方法提取样品，并使用液相色谱-质谱联用仪分析以测定 T1AM 及其代谢物，即 3-碘甲状腺乙酸（TA1）、甲状腺胺、甲状腺乙酸、N-乙酰-T1AM 和 T1AM 酯。
在含有 FBS 的缓冲液中，T1AM 随时间呈指数下降，半衰期为 6-17 分钟，具体取决于 FBS 含量，60 分钟后，T1AM 平均为基线的 0-10%。除了最低量的 TA1 外，未检测到 T1AM 代谢物。值得注意的是，d4-T1AM 的下降速度要低得多，在 60 分钟时达到基线的 50-70%。这些效应通过蛋白质变性完全消除，通过半卡巴肼部分消除。在存在细胞的情况下，T1AM 浓度在 60 分钟内几乎降至 0，但 TA1 在孵育培养基中积累，具有定量回收率。在大鼠器官匀浆和人血中，通过同位素差异证实了 T1AM 浓度的自发下降。
这些结果表明，血液和组织蛋白结合和隔离 T1AM 和/或其醛衍生物，具有显著的同位素效应。这些问题可能是导致分析内源性 T1AM 分析复杂化的技术问题的原因。
