Endocrine Abstracts

Thyroid hormone (T₃) is mostly produced in peripheral tissues by thyroxine (T₄) deiodination, and its functional effects are directly related to tissue concentration, which is quite difficult to assay by conventional immunological methods. We report a novel technique, based on tandem mass spectrometry (MS/MS) coupled to HPLC, which was used in left ventricular myocardium obtained from rat and human hearts.

Samples were homogenized in phosphate buffer (pH 7.4). After centrifugation, supernatants were spiked with stable-isotope-labelled internal standards (IS: ¹³C₆-T₃ and ¹³C₆-T₄) and extracted by SPE. Dried residues were reconstituted and incubated with 3.0 N HCl in n-butanol, obtaining the butyl esters of T₃, T₄, and ISs. After removing excess reagents, residues were reconstituted with methanol/HCl 0.1 M (50:50 v:v) and injected in the HPLC-MS/MS system (AB-Sciex API 4000). Calibration curves were built with standard solutions containing both analytes at concentrations ranging from 1 to 50 ng/ml and the same amount of IS as put in the samples.

Sample derivatization increased method sensitivity and accuracy, and the minimum amount of tissue needed was ~100–150 mg. In control rat myocardium, tissue T₃ and T₄ averaged 1.59±0.11 and 2.24±0.22 pmol/g respectively (corresponding plasma free T₃ and T₄ were 3.69±0.39 and 17.09±1.56 pM). In animals treated with low-dose (6 μg/kg per day) or high-dose (45 μg/kg per day) T₃, tissue T₃ increased to 3.12±0.29 and 6.76±1.37 pmol/g (plasma free T₃ was 8.06±0.83 and 19.59±3.80 pM), while tissue T₄ decreased to 0.79±0.06 and 0.77±0.02 pmol/g (plasma free T₄ was 6.34±1.41 and 3.49±0.43 pM). In human samples obtained from transplanted hearts T₃ and T₄ averaged 1.51±0.16 and 5.94±0.63 pmol/g.

In conclusion an HPLC-MS/MS method based on derivatisation with buthanol enables T₃ and T₄ assay in ≥150 mg myocardial samples. Tissue T₃ and T₄ assay may be critical to understand the role of thyroid hormones in physiological and pathophysiological conditions.