Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method

Abstract

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5-20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure.

Keywords: LC-MS/MS; automated SPE; brain; cerebellum; cortex; rat; thyroid hormones.

Figure 1

Each graph shows the concentration in rat brain at different ages of each analyte. Samples were derived from five studies, and age groups are differentiated by different symbols: triangles, PND4; squares, PND21; stars, 71–72 d; and dots, 97–99 d. The symbols are colored in blueish or reddish tones referring to male (blueish) or female (reddish) rats. In each box plot a “+” marks the mean of the data group. Notable is the increase in T4, T3 and T2 concentrations on PND21 compared to other age groups and the opposite concentration pattern over age of rT3.

Figure 2

TH analysis in specific rat brain regions on PND4 and PND22 and in 71–72-day-old rats. The Y axis shows concentration in ng/g and the x axis male and female rats for each age group. The light-gray bar demonstrates the concentration found in cerebellum and the dark-gray bar in cortex. On PND22, T4, T3 and T2 concentrations yielded a similar concentration in both brain regions, whereas rT3 concentration sharply decreased in cerebellum as compared to cortex. On PND4, T3 concentration in cerebellum was one-third of the concentration found in cortex. T2 was not detected in cerebellum on PND4 in female rats and only in three out of six male rat pups.

Figure 3

Comparison of TH concentration between male and female rats on PND4 and -21. Each graph shows concentration in male and female rat brain for each analyte and in each box plot the mean is marked with a “+”. In the presented data, the triangles depict PND4 and dots PND21; blueish color reflects male and reddish color female rats. The data were analyzed by unpaired Welch t-test to determine difference between male and female rats in each TH analyte concentration on PND4 and PND21, respectively. No statistically significant difference was observed.