Thyroid hormones (TH) of maternal origin are critical for the proper fetal development, especially during early pregnancy. Even minor changes in maternal TH circulation can lead to various adverse outcomes. Recent studies found that the metabolites of thyroxine (T4) also play an important physiological role. For example, 3,5-diiodo-L-thyronine (T2) and 3,3′-diiodo-L-thyronine (rT2) can suppress the thyroid stimulating hormone (TSH) level and increase the resting metabolic rate. 3-iodothyronamine (T1AM) administration in mice leads to a hypometabolic state. These metabolites may have influences on the fetus. Having the capacity to make a comprehensive analysis of T4 and the metabolites in placenta provides a diagnostic tool for the placental TH homeostasis. Routine TH assessment has long been achieved by measuring T4, triiodo-L-thyronine (T3), and TSH in blood using immunoassay (IA) method, which is of high sensitivity, but is prone to nonspecific interferences. Methods based on liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) showed better accuracy and reliability. In this study, we report a method for the determination of T4, T3, 3,3′,5′-triiodo-L-thyronine (rT3), T2, rT2, 3-iodo-L-thyronine (T1), and T1AM in the placenta. The method was optimized using isotope (13C-T4, 13C-T3, 13C-rT3, 13C-T2) dilution methodology and determined by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The calibration ranges from 0.5 to 150 ng/ml with R2 values > 0.99. The method detection limits (MDLs) and the method quantification limits (MQLs) were 0.010.2 ng/g and 0.040.7 ng/g, respectively. The spike-recoveries for THs (except for T1 and T1AM) were between 81.0% and 112%, with a coefficient of variation (CV) of 0.56.2%. The intra-day CVs and inter-day CVs were 0.5% 10.3% and 1.19% 8.88%, respectively. The method was adopted for TH measurement in human and mouse placenta. The concentrations of T4, T3, rT3, and T2 were 22.935.0 ng/g, 0.320.46 ng/g, 2.863.69 ng/g, and 0.160.26 ng/g in human placenta, and 2.053.51 ng/g, 0.370.62 ng/g, 0.961.3 ng/g, and 0.070.13 ng/g in mouse placenta, respectively. The presence of T2 was tracked in placenta tissue for the first time, indicating improved selectivity and sensitivity of our method. The validated method allows comprehensive evaluation of total T4 and metabolites in the placenta.
19 - 22 May 2018
European Society of Endocrinology