Background: 3,5-Diiodo-L-thyronine (3,5-T2), a potential metabolite of 3,3',5-triiodothyronine (T3), exerts marked metabolic actions without the undesirable cardiac and central side effects of T3. So far the lack of reliable quantification methods for endogenous 3,5-T2 in human serum has limited further insight into its physiological and pathophysiological roles in endocrine homeostasis and disease status.
Methods: Monoclonal anti-3,5-T2 antibodies (3,5-T2 mAbs) were produced in mice. We developed a competitive chemiluminescence immunoassay (CLIA) with one selected mAb and optimized it for high sensitivity, linearity, recovery, and low cross-reactivity to structurally related thyroid hormones (THs) and thyronamines. The CLIA was then used to investigate the origin and action of 3,5-T2 in humans under physiological and pathophysiological conditions in comparison with THs. Patient analysis included individuals with confirmed hypo- or hyperthyroidism and a separate population of thyroidectomized patients on L-thyroxine (T4) replacement therapy.
Results: 3,5-T2 is stable in human serum after storage at 4°C or room temperature as well as several freeze-thaw cycles. The immunoassay did not show any significant cross-reactivity with naturally occurring TH metabolites in physiological and pathophysiological concentrations. The assay shows a lower detection limit of 0.2 nM 3,5-T2 and an upper detection limit of 10.0 nM. The newly established CLIA generates reliable results after spiking exogenous 3,5-T2 or by linear dilution of sera. Intra-assay variation is between 4.1% and 9.0%. Overall mean of variation between different assays is 5.6%-12.9%. 3,5-T2 serum concentrations do not differ in hyperthyroid (0.31 ± 0.02 nM, n=24) compared to hypothyroid (0.43 ± 0.04 nM, n=31) individuals. 3,5-T2 was detectable and elevated in serum from thyroidectomized and T4-substituted patients (0.48 ± 0.03 nM, n=100) in comparison to a sex- and age-matched control group (0.29 ± 0.01 nM, n=99).
Conclusion: The established CLIA is highly specific, sensitive, precise and accurate for 3,5-T2 detection in human serum. Because 3,5-T2 is not regulated in conditions of an altered thyroid state, it is most likely that serum 3,5-T2 concentrations are not directly dependent on feedback regulation via the hypothalamic-pituitary axis. In addition 3,5-T2 is present in thyroidectomized individuals on T4 substitution, and it is elevated after T4 substitution compared with healthy controls. We conclude that these data support extrathyroidal production of 3,5-T2 from T4.