Extraction and quantification of thyroid hormones in selected regions and subcellular fractions of the rat brain

There is increasing evidence of an involvement of thyroid hormones in numerous physiological processes of the adult vertebrate brain. However, the only valid method available for measuring triiodothyronine (T3) in brain tissue is time-consuming and not sufficiently sensitive to determine hormone concentrations in small, but physiologically important areas such as the amygdala and septum. We therefore developed a protocol for reliable measurement of the concentrations of thyroxine (T4) and T3 in brain tissue. This was achieved by combining a new method of extracting iodothyronines with highly sensitive, accurate and reproducible radioimmunoassays (RIAs) in order to be able to detect T4 and T3 in homogenates and even subcellular fractions (nuclear, synaptosomal and mitochondrial) in up to 11 regions of the rat brain. The iodothyronines were extracted from tissue samples by adding 100% methanol containing 1 mM PTU. Recoveries of 72.8 +/- 5.5% and 83.2 +/- 3.3% for T4 and T3, respectively, were obtained. The RIA detection thresholds were 10 fmol/g for T4 and 18 fmol/g for T3. Only 0.2% of the antibody for T4 cross-reacted with T3 and 0.95% reverse T3. T3 antibody (0.05%) reacted with T4 and 0.01% with 3,5-T2. The T4 concentrations in the homogenates of selected areas of the brain ranged between 1 and 4 pmol/g, whereas those of T3 ranged between 0.5 and 4 pmol/g. The T3 levels ranged between 190 and 470 fmol/mg protein, 38 and 110 fmol/g protein and 25 and 180 fmol/mg protein in the nuclei, synaptosomes and mitochondriae, respectively. In conclusion, the newly developed method enabled us to determine both T4 and T3 concentrations in homogenates and T3 in subcellular fractions of regions of the brain as small as the septum and amygdala.