Detection of 3,5-Diiodothyronine in Sera of Patients with Altered Thyroid Status Using a New Monoclonal Antibody-Based Chemiluminescence Immunoassay

Background: 3,5-Diiodo-L-thyronine (3,5-T-2), a potential metabolite of 3,3',5-triiodothyronine (T-3), exerts marked metabolic actions without the undesirable cardiac and central side effects of T-3. So far the lack of reliable quantification methods for endogenous 3,5-T-2 in human serum has limited further insight into its physiological and pathophysiological roles in endocrine homeostasis and disease status. Methods: Monoclonal anti-3,5-T-2 antibodies (3,5-T-2 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-T-2 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 (T-4) replacement therapy. Results: 3,5-T-2 is stable in human serum after storage at 4 degrees 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-T-2 and an upper detection limit of 10.0 nM. The newly established CLIA generates reliable results after spiking exogenous 3,5-T-2 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-T-2 serum concentrations do not differ in hyperthyroid (0.31+/-0.02nM, n = 24) compared to hypothyroid (0.43+/-0.04nM, n = 31) individuals. 3,5-T-2 was detectable and elevated in serum from thyroidectomized and T-4-substituted patients (0.48+/-0.03nM, n = 100) in comparison to a sex-and age-matched control group (0.29+/-0.01nM, n = 99). Conclusion: The established CLIA is highly specific, sensitive, precise and accurate for 3,5-T-2 detection in human serum. Because 3,5-T-2 is not regulated in conditions of an altered thyroid state, it is most likely that serum 3,5-T-2 concentrations are not directly dependent on feedback regulation via the hypothalamic-pituitary axis. In addition 3,5-T-2 is present in thyroidectomized individuals on T-4 substitution, and it is elevated after T-4 substitution compared with healthy controls. We conclude that these data support extrathyroidal production of 3,5-T-2 from T-4.