Little is known about the kinetics and metabolism of thyroid hormones in the hypothyroid state. To investigate these factors, we developed a reliable method for measurement of serum thyroxine (T 4 ), triiodothyronine (T 3 ), reverse-T 3 (rT 3 ) and stable isotope-labeled T 4 ([ 13 C 9 ]T 4 ), using online solid-phase extraction liquid chromatography-mass spectrometry/mass spectrometry (online SPE LC-MS/MS). We measured supply and turnover rates of T 4 in thyroidectomized (Tx) rats using [ 13 C 9 ]T 4 as a tracer. In rats, serum T 4 , T 3 and rT 3 were decreased but not completely ablated after surgical Tx. Endogenous T 4 and T 3 levels in Tx rats were maintained at a constant low level throughout the experimental period. [ 13 C 9 ]T 4 levels declined with a half-life of w1 . 2 days after it was administered to Tx rats intravenously. These findings strongly suggest that serum T 4 levels in Tx rats are maintained by T 4 supplied by extra-thyroidal tissues (e.g. secretion of extra-thyroidal storage, enhancement of enterohepatic recirculation, and production in extra-thyroidal tissues). Moreover, the turnover rate of T 4 in Tx rats was approximately twofold lower than in controls. This finding suggests that degradation of serum T 4 is repressed by Tx. In conclusion, serum T 4 is maintained at a constant low level by T 4 supply from extra-thyroidal tissues and repression of T 4 degradation in Tx rats. The powerful online SPE LC-MS/MS tool can be used to investigate thyroid hormones kinetics and metabolism, and thus has the potential to be used as a diagnostic tool and to investigate the pathogenesis of thyroid disease.

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ObjectiveFor measuring serum 3,3′,5′-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3′-triiodothyronine (T3) levels.MethodsAssay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively.ResultsMatrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001).ConclusionsOur LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.

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