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

Authors
Journal
Brain Research Protocols
1385-299X
Publisher
Elsevier
Publication Date
Volume
4
Issue
1
Identifiers
DOI: 10.1016/s1385-299x(98)00056-7
Keywords
  • Thyroid Hormone
  • Iodothyronine Extraction
  • Ria
  • Radio-Labeling
  • Subcellular Fractions
Disciplines
  • Biology
  • Medicine

Abstract

Abstract 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 (T 3) 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 (T 4) and T 3 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 T 4 and T 3 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 T 4 and T 3, respectively, were obtained. The RIA detection thresholds were 10 fmol/g for T 4 and 18 fmol/g for T 3. Only 0.2% of the antibody for T 4 cross-reacted with T 3 and 0.95% reverse T 3. T 3 antibody (0.05%) reacted with T 4 and 0.01% with 3,5-T 2. The T 4 concentrations in the homogenates of selected areas of the brain ranged between 1 and 4 pmol/g, whereas those of T 3 ranged between 0.5 and 4 pmol/g. The T 3 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 T 4 and T 3 concentrations in homogenates and T 3 in subcellular fractions of regions of the brain as small as the septum and amygdala. Themes: Endocrine and autonomic regulation Topics: Neuroendocrine regulation: other

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